Toxoplasma gondii vaccines and their use

ABSTRACT

Disclosed herein are polynucleotides encoding multi-epitope polypeptides and assemblies thereof, and their use for treating or limiting  Toxoplasma gondii  infection.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/324,225 filed Apr. 18, 2016, incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under grant numbersDMID-NIAID U01 AI77887, R01 27530, and U19 A1110819, all awarded by TheNational Institutes of Health. The U.S. Government has certain rights inthe invention.

BACKGROUND

Toxoplasma gondii is an intracellular parasite that can cause severeocular and neurological diseases in fetuses, newborn infants, andimmunocompromised individuals (1). The acute infection is characterizedby proliferation of tachyzoites, which replicate rapidly within hostcells and lyse their host cells within 24-48 hours to release largenumbers of progeny. In response to immune pressure, the parasitedifferentiates into a slow-growing form called bradyzoites, whichresides within intracellular cysts. Formation of tissue cysts normallyoccurs in long-lived cells such as muscle or neuronal cells. Althoughantiparasitic medicines such as sulfadiazine and pyrimethamine areeffective against tachyzoites, they are associated with toxicity orhypersensitivity and do not eliminate the latent, cyst form of theparasite. Thus, there is a need for development of a safe, protectivevaccine.

SUMMARY OF THE INVENTION

In one aspect, the invention provides isolated polynucleotides encodinga chimeric polypeptide, wherein the chimeric polypeptide comprises

(a) a plurality of first peptide domains wherein each first peptidedomain is a CD8⁺T cell eliciting epitope, including but not limited toFLSLSLLVI (SEQ ID NO: 1), FMIAFISCFA (SEQ ID NO: 2), FVIFACNFV (SEQ IDNO: 3), FMIVSISLV (SEQ ID NO: 4), FLLGLLVHV (SEQ ID NO: 5), FLTDYIPGA(SEQ ID NO: 6), ITMGSLFFV (SEQ ID NO: 7), GLAAAVVAV (SEQ ID NO: 8),VLLPVLFGV (SEQ ID NO: 9), FAAAFFPAV (SEQ ID NO: 10), VVFVVFMGV (SEQ IDNO: 11), FMGVLVNSL (SEQ ID NO: 12), FLVPFVVFL (SEQ ID NO: 13), STFWPCLLR(SEQ ID NO: 14), SSAYVFSVK (SEQ ID NO: 15), KSFKDILPK (SEQ ID NO: 16),AVVSLLRLLK (SEQ ID NO: 17), and AMLTAFFLR (SEQ ID NO: 18)); and

(b) one or more second peptide domains, wherein each second peptidedomain is a CD4+ epitope, including but not limited to AKFVAAWTLKAAA(SEQ ID NO: 19), AVEIHRPVPGTAPPS (SEQ ID NO: 20), IRLLASLHH (SEQ ID NO:39), LIRLLASLH (SEQ ID NO: 40), LTLQLIRLL (SEQ ID NO: 41, VIEEFNRI (SEQID NO: 42), LQLIRLLAS (SEQ ID NO: 43), IDVVIEELF (SEQ ID NO: 44), or aderivative thereof.

In one embodiment, the plurality of first peptide domains comprises atleast 5 peptide domains, wherein the at least 5 peptide domains includeKSFKDILPK (SEQ ID NO: 16), STFWPCLLR (SEQ ID NO: 14), AVVSLLRLLK (SEQ IDNO: 17), SSAYVFSVK (SEQ ID NO: 15), AMLTAFFLR (SEQ ID NO: 18). Inanother embodiment, the one or more second peptide domains includeAKFVAAWTLKAAA (SEQ ID NO: 19) or a derivative thereof. In anotherembodiment, the chimeric polypeptide comprises the amino acid sequenceAVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLR (SEQ ID NO: 24). In another embodiment, the chimericpolypeptide further comprises a third peptide domain comprising apeptide capable of promoting self-assembly/multimerization (2, 3, 4, 5,6, or more assembled copies) of the polypeptide. In further embodiments,the chimeric polypeptide comprises the amino acid sequence SE IDNOS:27-28.

In another aspect, the invention provides recombinant expressionvectors, comprising the isolated polynucleotide of any embodiment orcombination of embodiments of the invention operatively linked to acontrol sequence. In a further embodiment, the invention provideschimeric polypeptides comprising the chimeric polypeptide encoded by thepolynucleotide or the expression vector of any embodiment or combinationof embodiment of the claims. In a further aspect, the invention providesassemblies comprising a plurality of the polypeptides or RNAs of theinvention.

The invention further provides pharmaceutical composition, comprising:(a) the chimeric polynucleotide, the expression vector, the chimericpolypeptide, the chimeric RNA, and/or the assembly of any embodiment orcombination of embodiments of the invention; and

(b) a pharmaceutically acceptable carrier. In one embodiment, thecomposition comprises an adjuvant, such as a TLR4 ligand or glucoglucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE).

In another aspect, the invention provides methods for treating orlimiting Toxoplasma gondii infection, comprising administering to asubject at risk of Toxoplasma gondii infection an amount effective totreat or limit the infection of the chimeric polynucleotide, theexpression vector, the chimeric polypeptide, the chimeric RNA, and/orthe assembly of any embodiment or combination of embodiments of theinvention.

DESCRIPTION OF THE FIGURES

FIG. 1. Testing of peptides with PBMCs from HLA-A03 supertype T. gondiiseropositive and seronegative donors. (A) PBMC from donors who wereseropositive and seronegative for T. gondii were tested for response topredicted HLA-A03-restricted CD8+ T cell epitopes. Individual peptideswere tested using IFN-γ ELISpot assay. (B) Concanavalin A (Con A) andtachyzoite antigen lysates (TLA) were used as controls. In A,experiments were performed 3 times. A representative experiment with oneseropositive and one seronegative person shows the variability for eachindividual. For each person for each peptide, there were 6determinations (wells). Each symbol represents one of these measurementsof IFN-γ. The horizontal line is the mean of these 6 determinations withthe SD shown. In B, methods, numbers of determinations, and comparisonswere the same as for the peptides but were for Con A and TLA stimulationas controls. In 3 replicate experiments, PBMCs also were obtained from 3T. gondii seropositive and 3 T. gondii seronegative HLA-A03 individuals.In these experiments, in the comparison of 3 seropositive and 3seronegative persons, differences between the seropositive andseronegative persons were significant for each peptide when tested byStudent's t test (P<0.05; n=3 per group, data not shown). Stimulationfor seropositive and seronegative persons for TLA were different andachieved statistical significance (P<0.05). In contrast, stimulationwith Con A demonstrated response of PBMC from both seronegative andseropositive donors (data not shown).

FIG. 2. Immunogenicity and efficacy of 5 identified A11-restricted CD8+T cell epitope pool peptides in combination with PADRE and GLA-SEadjuvant in HLA-A*11:01 transgenic mice. (A) T. gondii brain cystsnumber was significantly reduced in HLA-A*11:01 mice immunized with poolof peptides plus PADRE and GLA-SE adjuvant at 21 days after challengewith 2,000 T. gondii ME49-Fluc (Type II) parasites (n=8 mice per group,in 2 replicate experiments with 4 mice per experiment, pooled; *P<0.013,Student's t test). (B) Quantitative PCR for the parasite burden in theT. gondii challenged HLA-A*11:01 mice brains (n=7 mice per groupcombining 2 replicate experiments, *P<0.004, Student's t test). (C andD) Flow cytometry gating for CD8+ memory T cells 11 weeks afterimmunization of HLA-A*11:01 mice with pooled adjuvanted peptides. Cellsare gated on CD3+CD8+ T cells. Memory T cells were defined asCD44hiCD45RBlo. For each group, a representative FACS plot is shown withthe percent of CD8+ memory T cells shown. For each group, n=3 anddifferences were significant; P<0.05, Student's t test. This experimentis representative of 2 replicates. Horizontal lines are means and SDsare shown in D.

FIG. 3. Schematic diagram of the DNA vaccine construct. (A and B) Theorientation of the HLA-A*11:01-restricted CD8+ T cell epitopes and PADREin the synthetic gene is shown with 2 different types of spacers, calledLO and AZ, for N/KAAA and GPGPG (SEQ ID NO: 23) linker, respectively.(C) SDS-PAGE 4%-20% of the purified LO and AZ proteins.

FIG. 4. LO and AZ elicit specific immune responses in HLA-A03seropositive and seronegative donors. (A-C) ELISpot showing IFN-γ spotformation. PBMCs were tested using LO, AZ, and pool of peptides. (C) ConA and TLA were used as controls. Variability between determinations forsingle donors are shown in panels A and B with n=6 determinations (asdescribed in FIG. 1). In A and B, the data shown are a singlerepresentative experiment from one T. gondii seropositive and one T.gondii seronegative HLA-A03-supertype donor. Each experiment was carriedout with 6 determinations. Replicate experiments also were performed 3times with PBMCs from 3 T. gondii seropositive and 3 T. gondiiseronegative HLA-A03 individuals. Two-tailed Student's t test was usedfor statistical analysis comparing differences between the 2 groups (n=3per group, P<0.05). One-way ANOVA was performed before the Student's ttest to determine whether there was an overall difference between thegroups. Differences between the stimulation of seropositive andseronegative individuals' PBMCs by the poly-epitope proteins weresignificant (P<0.05, data not shown). Similarly, TLA stimulated theseropositive persons' PBMCs, but Con A did not (data not shown).

FIG. 5. Multivalent polypeptide LO and AZ protective efficacy in vivowith HLA-A*11:01 transgenic mice survival curve after challenge withType II parasites. Two weeks after last immunization, the transgenicmice immunized with pooled LO and AZ proteins (prot) in combination witheither adjuvant GLA-SE or ALUM adjuvant or injected with PBS wereinfected with 2,000 Me49 (Fluc) parasites. The survival rates of the 2groups were recorded. This figure shows data from mice in both of 2replicate experiments combined (n=5 control and 5 immunized mice).Kaplan-Meier curves were generated and survival compared across groupsusing the log-rank test, P<0.05. Differences between protLO+AZ+GLA-SEand all other groups were significant (P<0.05) and differences betweenprotLO+AZ+GLA-SE and protLO+AZ+ALUM also were compared for survivalusing log-rank test and Kaplan-Meier analysis.

FIG. 6. DNA prime-protein boost regimen. (A) pMB75.6 vector used as aDNA vaccine vector for this study. Lane P: pMB75.6 plasmid; lane 2:pMB75.6 plasmid digested with EcoRI and BglII; lane M: KB ladder. (B) T.gondii brain cysts luciferase expression was reduced in HLA-A*11:01 miceimmunized with DNA/protein boost at 21 days after challenge with 2,000T. gondii ME49-Fluc (Type II) expressing luciferase. (C) Xenogen imagingof brain ex vivo following the injection of luciferin into theretro-orbital plexus and then exposure of the brain to luciferinsolution. n=5 per group, *P=0.0008, **P=0.004, ***P=0.004 (Student's ttest after one-way ANOVA). (D) Enumeration of cysts was performed withbrains of mice challenged 21 days after final immunization. Theseexperiments were performed at least 2 times, and one representativeexperiment of 2 is shown: n=5 control and 5 immunized mice. *P=0.002,**P=0.003, ***P=0.004; Student's t test was used to compare the groups.For C and D, the plots show median, with box extending from the 25th to75th percentile and the whiskers extending from minimum and maximumvalues of the data set. One-way ANOVA was performed before the Student'st test to determine whether there was an overall difference between thegroups.

FIG. 7. CD8+ T cell responses in HLA-A*11:01 mice followingimmunization. (A) Splenocytes from immunized mice with LO DNA, AZ DNA,and multi-epitope polypeptides alone or combined were harvested for10-14 days after immunization and exposed to LO or AZ polypeptide for exvivo IFN-γ expression. Quantitation of IFN-γ from mouse splenocytes wasevaluated in 2 separate experiments. Each experiment (n=4 mice) wasevaluated for comparison for vaccination with AZ protein compared withcontrol and for LO protein compared with control; significance evaluatedwith Student's t test. (B) HLA-A*11:01 transgenic mice survival curveafter challenge with Type II parasites. Two weeks after lastimmunization, the transgenic mice immunized with empty vector, LO DNA+LOpolypeptide, or LO polypeptide, or were injected with PBS were infectedwith 2,000 T. gondii ME49-Fluc (Type II) parasites. The survival ratesof the 2 groups were recorded. Mice vaccinated with either LO proteinalone or LO DNA+LO protein were compared with control mice (PBS or emptyvector). Kaplan-Meier curves were generated and survival compared acrossgroups using the log-rank test, P<0.05. (n=8 mice per group in 2replicate experiments with 4 mice, shown pooled). (C and D) CD8+ memoryT cells. Flow cytometry gating for CD8+ memory T cells. Spleen cells aregated on CD3+CD8+ T cells. Memory T cells were defined asCD44hiCD45RBlo. For each group, a representative FACS plot is shown withthe percent of CD8+ memory T cells shown. All mouse experiments wererepeated at least twice (=2-4 mice in each group). *P<0.001, **P<0.001.In A and D; one-way ANOVA was performed before the Student's t test todetermine whether there was an overall difference between the groups.

FIG. 8. Multi-epitopes adjuvanted with GLA-SE are captured and presentedby MHC molecules on the APCs to T lymphocytes. (A) HLA-A*11:01transgenic mice immunized with LO protein plus GLA-SE were protectedcompared with control mice inoculated with PBS when they were challengedwith 20,000 T. gondii prugneaud strain (Fluc) luciferase expressingparasites after 4 and 6 days. (n=4 mice per group, 2 replicateexperiments.) (B) Assay demonstrating that GLA-SE is a TLR4 ligand thatleads to production of IL-6, IL-12, and TNF-α by PBMC. Stimulation ofhuman whole blood with GLA-SE. Heparinized whole blood was collectedfrom 6 healthy donors, and 200 μl was stimulated with 5 μg GLA-SE in96-well plates at 37° C. CO2. After 24 hours, plasma was removed andassayed for IL-6, IL-12(p40), and TNF-α by a custom Luminex-basedmultiplex immunoassay kit (Affymetrix eBioscience). Data were analyzedusing the Masterplex QT software (Miraibio). The cytokine productionstimulated by adjuvant was statistically significant for IL-6, IL-12,and TNF-α (P<0.05) compared with the unstimulated groups as assessed bythe Mann-Whitney U test (GraphPad Prism software). The plots showmedian, with box extending from the 25th to 75th percentile and thewhiskers extending from minimum and maximum values of the data set. (C)Multi-epitope proteins with GLA-SE are captured by theAntigen-presenting cells (APCs), and the peptides contained arepresented by MHC molecules on the APCs to T lymphocytes in both a classI and a class II pathway. This demonstrates that cross presentation intoa class I pathway must occur by virtue of effector function. APCs arealso activated through recognition of GLA-SE by TLR4 receptorsmolecules. This activation leads to the production of pro-inflammatorycytokines (IL-12, IL-6, TNF-α) and the expression of costimulatorymolecules on the cell surface.

FIG. 9: CD8-SAPNs elicit restricted CD8⁺ T and CD4⁺ T cellpeptide-specific immune response. ELISpot showing IFN-γ spot formation.Mouse splenocytes from Empty-SAPN, CD8-SAPN, and CD8-SAPN+GLA weretested using GRA6 peptide (GRA6₁₆₄₋₁₇₂) or PADRE. All peptides elicitedIFN-γ (p<0.05) compared to unstimulated cultures. Pooled peptidesappeared additive. The greatest effect occurred with the polypeptide asalso occurred in earlier studies²³. *=p<0.05.

FIG. 10: CD8-SAPNs are potent inducers of cell-mediated immune. a) IFN-γELISpot assay stimulated with a group of 5 peptides HLA-A*1101. b) Graphshows the count of spots for splenocytes of untreated, Empty-SAPN+GLA1CD8-SAPN+GLA group of mice. *=p<0.05 for all IFN-γ ELISpots compared tocontrols.

FIG. 11: SeaPorter TLR5 cell-line was exposed to varying concentrationsof each indicated protein, and the level of TLR5 stimulation wasdetermined by the level of SEAP expression. Fold increase in SEAPexpression for each protein sample over non-treated controls, error barsare standard error of the means. A two-way ANOVA model was fit withprotein concentration and type as factors. There was a significantprotein concentration by type interaction (p<0.001); this indicated thatthe differences across types depended on the concentration and that thedifferences across concentrations varied by type. Specifically, thereweren't statistically significant differences across types at the twolowest concentrations (0.01 and 0.1), but there were significantdifferences between types at the 1, 10, 100, and 1000 ng/mLconcentrations (p<0.001 for all). Subsequent pairwise contrasts at these4 concentrations found that the 5A11 Restricted CD8+ group (therecombinant protein without flagellin) was significantly different fromthe other 3 groups in all cases except for the 5A11 Restricted CD8+ vs.CD8-SAPN comparison at the 1 ng/mL concentration. In addition, at eachof these 4 concentrations, the Empty-SAPN was significantly different(greater than) from the CD8-SAPN. And as expected, there was asignificant concentration effect for all protein types (p<0.001) except5A11 Restricted CD8+ (p>0.9).

FIG. 12: T. gondii brain cysts luciferase expression was significantlyreduced in immunized HLA-A*1101 mice. HLA-A*1101 transgenic mice wereimmunized with GLA-SE adjuvanted Empty-SAPN or CD8-SAPN three times atintervals of two weeks. PBS was used as control. a) T. gondii braincysts luciferase expression was significantly reduced in HLA-A*1101 miceimmunized with CD8-SAPN plus GLA boost at 21 days after challenge with2000 Me49 (Fluc) T. gondii expressing luciferase. b) Xenogen imaging ofbrain ex vivo following the injection of luciferin into theretro-orbital plexus and then exposure of the brain to luciferinsolution. This figure shows data from mice in one of the replicateexperiments (n=4 control and 4 immunized mice). c) Enumeration of cystwas performed with brains of mice challenged 21 days after finalimmunization. SAPN reduced cyst numbers and luminescence (p<0.05).

FIG. 13: (A) Immunization with replicon elicits CD8 T splenocytes thatrespond to the immunizing CD8 T cell elicity peptides. Interferon gammaelisspot shows that response. (B) Histogram showing amounts ofinterferon gamma produced by immunization with the Toxoplasma gondiisequence in the replicon. These immunizations were protective (data notshown).

FIG. 14: Interferon gamma production by CD4 nd CD8 T cells. A.Interferon gamma production per CD4+T cell. Note that there is the mostproduction in G3 CD4 peptide AS15 plus GLA-SE where when PADRE ispresent this is diminished. B. Interferon gamma production per CD8+ Tcells. Note the group that contains CD9 peptides produces the mostinterferon gamma.

FIG. 15: Protection by immunization with AS15. A. AS15=CD4 plusadjuvant(GLA-SE) confers the greets protection measured as the leastfluorescence by the RH tachyzoite challenge. B. Luminescence in brain.AS15(CD4sp) protects against parasite burden luminescence. The liveparasite that is attenuated, delta RPS13 is robustly protective as aninternal control. All are significantly less than PBS control. C.Challenge with Me49 strain of T.gondii, 10 days after the secondimmunization. These mice have been immunized (or received PBS oradjuvant alone). The most robust protection was following immunizationwith AS15=CD4 peptide which is from T.gondii (diamonds and X).

FIG. 16: HLA A-11-restricted CD8⁺ T cell epitope peptides induce CD8+Memory T cells.

FIG. 17A: Schematic diagram of the A11 SAPN (SEQ ID NO:27) in monomeric,trimeric, pentameric, and particle form.

FIG. 17B: Schematic drawing of the ToxAll SAPN (SEQ ID NO:28) inmonomeric, trimeric, pentameric, and particle form.

FIG. 17C: Predicted and actual proper folding and size of ToxAll. (A)Western blot; (B) Electron micrograph; (C) Size Distribution by volume.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.),

“Guide to Protein Purification” in Methods in Enzymology (M. P.Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide toMethods and Applications (Innis, et al. 1990. Academic Press, San Diego,Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed.(R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin,Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While the specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

In a first aspect, the invention provides isolated polynucleotideencoding a chimeric polypeptide, wherein the chimeric polypeptidecomprises

(a) a plurality (2, 3, 4, 5, 6, 7, 8, 9, or more) of first peptidedomains wherein each first peptide domain is a CD8⁺ T cell elicitingepitope, including but not limited to FLSLSLLVI (SEQ ID NO: 1),FMIAFISCFA (SEQ ID NO: 2), FVIFACNFV (SEQ ID NO: 3), FMIVSISLV (SEQ IDNO: 4), FLLGLLVHV (SEQ ID NO: 5), FLTDYIPGA (SEQ ID NO: 6), ITMGSLFFV(SEQ ID NO: 7), GLAAAVVAV (SEQ ID NO: 8), VLLPVLFGV (SEQ ID NO: 9),FAAAFFPAV (SEQ ID NO: 10), VVFVVFMGV (SEQ ID NO: 11), FMGVLVNSL (SEQ IDNO: 12), FLVPFVVFL (SEQ ID NO: 13), STFWPCLLR (SEQ ID NO: 14), SSAYVFSVK(SEQ ID NO: 15), KSFKDILPK (SEQ ID NO: 16), AVVSLLRLLK (SEQ ID NO: 17),and AMLTAFFLR (SEQ ID NO: 18); and

(b) one or more second peptide domains, wherein each second peptidedomain is a CD4+ epitope, including but not limited to AKFVAAWTLKAAA(SEQ ID NO: 19), AVEIHRPVPGTAPPS (SEQ ID NO: 20), IRLLASLHH (SEQ ID NO:39), LIRLLASLH (SEQ ID NO: 40), LTLQLIRLL (SEQ ID NO: 41), VIEEFNRI (SEQID NO: 42), LQLIRLLAS (SEQ ID NO: 43), IDVVIEELF (SEQ ID NO: 44), or aderivative thereof.

The isolated polynucleotides of the invention are shown in the examplesthat follow to be useful, for example, as multi-epitope nucleic acidvaccines, or to encode multi-epitope protein vaccines against Toxoplasmagondii. These multi-epitope vaccines are shown to increase memory CD8⁺ Tcells that produced IFN-γ and to protects mice against parasite burdenwhen challenged with T. gondii, demonstrating their efficacy in crosspresentation of CD8⁺ T cell eliciting epitopes in a vaccine that canlimit or prevent toxoplasmosis.

The plurality of epitopes includes at least two different CD8⁺ T celleliciting epitopes. In one embodiment, the plurality of epitopesincludes 2, 3, 4, 5, 6, 7, 8, 9, or more of FLSLSLLVI (SEQ ID NO: 1),FMIAFISCFA (SEQ ID NO: 2), FVIFACNFV (SEQ ID NO: 3), FMIVSISLV (SEQ IDNO: 4), FLLGLLVHV (SEQ ID NO: 5), FLTDYIPGA (SEQ ID NO: 6), ITMGSLFFV(SEQ ID NO: 7), GLAAAVVAV (SEQ ID NO: 8), VLLPVLFGV (SEQ ID NO: 9),FAAAFFPAV (SEQ ID NO: 10), VVFVVFMGV (SEQ ID NO: 11), FMGVLVNSL (SEQ IDNO: 12), FLVPFVVFL (SEQ ID NO: 13), STFWPCLLR (SEQ ID NO: 14), SSAYVFSVK(SEQ ID NO: 15), KSFKDILPK (SEQ ID NO: 16), AVVSLLRLLK (SEQ ID NO: 17),and AMLTAFFLR (SEQ ID NO: 18). In one specific embodiment, the pluralityof first peptide domains comprises at least 5 peptide domains, whereinthe at least 5 peptide domains include KSFKDILPK (SEQ ID NO: 16),STFWPCLLR (SEQ ID NO: 14), AVVSLLRLLK (SEQ ID NO: 17), SSAYVFSVK (SEQ IDNO: 15), AMLTAFFLR (SEQ ID NO: 18). This embodiment is specificallydescribed in the examples that follow.

The second peptide domain may be present in one or more copies. Inanother specific embodiment, the one or more second peptide domainsinclude AKFVAAWTLKAAA (SEQ ID NO: 19) or a derivative thereof. Thisembodiment is also specifically described in the examples that follow.

In a further embodiment, some (2, 3, 4, 5, etc.) or all of the pluralityof first peptide domains are separated from the other first peptidedomains peptides by a spacer of 1 or more (2, 3, 4, 5, etc.) amino acidresidues and/or by the one or more second peptide domains. Wheremultiple spacers are included in a construct, each copy of the spacermay have the same sequence, or the spacers may include 2 or moredifferent spacers. In various specific embodiments, the spacer(s) is/areselected from the group consisting of N, K, NA₁₋₃ (SEQ ID NO: 21),KA₁₋₃(SEQ ID NO: 22), and/or GPGPG (SEQ ID NO: 23).

In one specific embodiment, the chimeric polypeptide comprises the aminoacid sequence

(SEQ ID NO: 24) AVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLR

In another embodiment, the chimeric polypeptide further comprises athird peptide domain comprising a peptide capable of promotingself-assembly/multimerization (2, 3, 4, 5, 6, or more assembled copies)of the polypeptide. In these embodiments, the polypeptides areself-assembling protein nanoparticles, as fully described in theexamples that follow. Any suitable peptide domain capable of promotingself-assembly/multimerization can be used. Non-limiting examples of thethird peptide domains known to those of skill in the art and suitablefor use in the present invention include, but are not limited to peptidehelices containing at least one helix, or a structure formed by a helix,a coil and another helix, etc., coiled coil structures, dimerizationdomains within, for example, many cell surface signaling receptors, Fcregions or hinge regions of an antibody, leucine zippers, the STATprotein N terminal domain, FK506 binding protein, the LexA proteinC-terminal domain, nuclear receptors, the FkpA N-terminal domain, orangecarotenoid protein from A. maxima, M1 matrix protein from influenza,neuraminidase from influenza virus, E. coli fuculose aldolase; and thelike. (see, e.g., O'Shea, Science. 254: 539 (1991), Barahmand-Pour etal., Curr. Top. Microbiol. Immunol. 211: 121-128 (1996); Klemm et al.,Annu. Rev. Immunol. 16: 569-592 (1998); Klemm et al., Annu. Rev.Immunol. 16: 569-592 (1998); Ho et al., Nature. 382: 822-826 (1996); andPomeranz et al., Biochem. 37: 965 (1998)). Further examples includeresidues 325 to 410 in the bovine papillomavirus E2 protein, (Dostatni,N., et al., EMBO J 7 (1988) 3807-3816; Haugen, T., et al. EMBO J 7(1988) 4245-4253; McBride, A., et al., EMBO J 7 (1988) 533-539; McBride,A., et al., Proc Natl Acad Sci USA 86 (1989) 510-514),Type I deiodinase(D1): DFLVIYIEEAHASDGW (SEQ ID NO: 31) or ADFL-YI-EAH-DGW (SEQ ID NO:32); HIV-1 Capsid Protein: QGPKEPFRDYVDRFYKTLRA (SEQ ID NO: 33); leucinezipper dimerization motif of yeast GCN4: HMKQL D VEEL S NYHL N VARL KVGER (SEQ ID NO: 34); leucine zipper in Escherichia coli transcriptionalantiterminator protein; and BglG: GVTQLMREMLQLIKFQFSLNYQEESLSYQRLVT (SEQID NO: 35). In various specific embodiments, the third peptide domaincomprises one or more copies of a pentameric coiled coil (such asWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWM (SEQ ID NO: 25)), a trimericcoiled coil such as RLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEEL(SEQ ID NO: 26), or both.

In another embodiment, the third polypeptide comprises a flagellinprotein or domain thereof, including but not limited to:

(SEQ ID NO: 45) MAQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNV; and/or (SEQ ID NO: 46)TENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLRof Salmonella enterica flagellin.

In another specific embodiment, the chimeric polypeptide comprises orconsists of the amino acid sequence selected from the group consistingof SEQ ID NOS:27-28. A11 (residues in parentheses are optional)

(SEQ ID NO: 27) ((MGDKHHHHHHHHHH))KDGSDKGSWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWMGGRLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEELARGMAQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNVQQEYESDDAVVSLLRLLKNAMLTAFFLRNAAA KSFKDILPK KAAA

KAAAK FVAAWTLKAAAKSTFWPCLLRDSDSDTENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTS VLAQANQVPQNVLSLLR;and

Tox-all (Residues in Parentheses are Optional)

(SEQ ID NO: 28) (MGDDHHHHHHHHHH)WFMGVLVNSLQDITMGSLFFVQDFMIVSISLVQDGLAAAVVAVQDLPQFATAATRDSPASGRYIQQMLDQRCQEIAAELCQSGLRKMCVPSSRIVARNAVGITHQNTLQWRCFDTASLLESNQENNGVNCVDDCGHTIPCPGGVHRQNSNHATRHEILSKLVEEGVQRFCSPYQASANKYCNDKFPGTIARRSKGFGNNVEVAWRCYEKASLLYSVYAECASNCGTTWYCPGGRRGTSTELDKRHYTEEEGIRQAIGSVDSPCSEVEVCLPKDENPPLCLDESGQISRGSWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWMGGRLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEELARGMAQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNVQQEYESDDAVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLRDSDSDTENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLL R.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine(Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q),glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

As used throughout the present application, the term “polypeptide” isused in its broadest sense to refer to a sequence of subunit aminoacids. The polypeptides of the invention may comprise L-amino acids,D-amino acids (which are resistant to L-amino acid-specific proteases invivo), or a combination of D- and L-amino acids. The polypeptidesdescribed herein may be chemically synthesized or recombinantlyexpressed. The polypeptides may be linked to other compounds to promotean increased half-life in vivo, such as by PEGylation, HESylation,PASylation, glycosylation, or may be produced as an Fc-fusion or inde-immunized variants. Such linkage can be covalent or non-covalent asis understood by those of skill in the art.

In another embodiment, the chimeric polypeptide further comprises asecretory signal. Any suitable secretory signal may be used; in onenon-limiting embodiment, the secretory signal is the murine Igκ-chainsignal sequence (MGMQVQIQSLFLLLLWVPGSRG (SEQ ID NO: 47)). In a preferredembodiment, the secretory signal is present at the N-terminus of thechimeric polypeptide.

In another aspect, the invention provides recombinant expression vector,comprising the isolated polynucleotide of any embodiment or combinationof embodiments of the invention operatively linked to a controlsequence.

As used herein, a “recombinant expression vector” includes vectors thatoperatively link a nucleic acid coding region or gene to any controlsequences capable of effecting expression of the encoded polypeptide.“Control sequences” operably linked to the nucleic acid sequences of theinvention are nucleic acid sequences capable of effecting the expressionof the nucleic acid molecules. The control sequences need not becontiguous with the nucleic acid sequences, so long as they function todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the nucleic acid sequences and the promoter sequence canstill be considered “operably linked” to the coding sequence. Other suchcontrol sequences include, but are not limited to, polyadenylationsignals, termination signals, and ribosome binding sites. Suchexpression vectors can be of any type known in the art, including butnot limited plasmid and viral-based expression vectors. The controlsequence used to drive expression of the disclosed nucleic acidsequences in a mammalian system may be constitutive (driven by any of avariety of promoters, including but not limited to, CMV, SV40, RSV,actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive). The construction of expression vectors for use intransfecting prokaryotic cells is also well known in the art, and thuscan be accomplished via standard techniques. (See, for example,Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin,Tex.). The expression vector must be replicable in the host organismseither as an episome or by integration into host chromosomal DNA. In apreferred embodiment, the expression vector comprises a plasmid.However, the invention is intended to include other expression vectorsthat serve equivalent functions, such as viral vectors.

In one embodiment, of the recombinant expression vectors of theinvention, the control sequences comprise viral proteins required forRNA replication, such as viral proteins encoded by an alphavirus genomeincluding but not limited to Venezuelan Equine Encephalitis (VEE)alphavirus. In a further embodiment, the vector comprises a geneencoding a self-amplifying RNA operatively linked to the polynucleotide(i.e.: self-amplification of the self-amplifying RNA results inamplification of RNA encoding the chimeric polypeptide). Any suitableself-amplifying RNA may be used, including but not limited to theself-amplifying RNA of SEQ ID NO:29.

(SEQ ID NO: 29) GAAUUUGCUGCGACGAUGGGUAUGCAAGUCCAGAUCCAGAGCCUGUUCCUGCUGCUGCUGUGGGUGCCGGGUUCACGCGGUAUGGCGGUGGUUAGCCUGCUGCGUCUGCUGAAAAACGCCAUGCUGACCGCAUUUUUCCUGCGCAAUGCGGCCGCAAAGAGUUUCAAGGAUAUCCUGCCGAAAAAGGCUGCGGCCAGCUCUGCGUAUGUCUUUUCCGUGAAAAAGGCAGCUGCGAAAUUCGUUGCUGCCUGGACCCUGAAAGCUGCCGCUAAAUCGACGUUCUGGCCGUGUCUGCUGCG U.

In another embodiment, the self-amplifying RNA is encoded by the nucleicacid sequence of SEQ ID NO:30.

(SEQ ID NO: 30) CATGGGTATGCAGGTCCAGATTCAGTCACTCTTTCTCCTCCTCCTCTGGGTCCCCGGTAGCCGGGGTATGGCCGTGGTCAGCCTGCTCAGGCTGCTCAAGAACGCCATGCTGACCGCTTTCTTTCTCAGAAATGCCGCTGCAAAGTCTTTCAAAGACATCCTGCCCAAGAAAGCCGCTGCAAGCTCCGCCTACGTGTTCAGTGTCAAGAAAGCCGCTGCAAAATTTGTGGCC

In another aspect, the invention provides chimeric polypeptidescomprising a polypeptide encoded by the polynucleotide or expressionvector of any embodiment or combination of the embodiments of thepresent invention, or a chimeric RNA comprising an RNA encoded by thepolynucleotide or expression vector of any embodiment or combination ofthe embodiments of the present invention.

In a further aspect, the invention provides assemblies, comprising aplurality (2, 3, 4, 5, 6, or more) of the polypeptides or RNAs of anyembodiment or combination of embodiments of the invention having thethird peptide domain. In this embodiment, the plurality of polypeptidesare self-assembled via non-covalent binding of the third domains. Anysuitable peptide domain capable of promotingself-assembly/multimerization can be used. Non-limiting examples of thethird peptide domains known to those of skill in the art and suitablefor use in the present invention include, but are not limited to peptidehelices containing at least one helix, or a structure formed by a helix,a coil and another helix, etc., coiled coil structures, dimerizationdomains within, for example, many cell surface signaling receptors, Fcregions or hinge regions of an antibody, leucine zippers, the STATprotein N terminal domain, FK506 binding protein, the LexA proteinC-terminal domain, nuclear receptors, the FkpA N-terminal domain, orangecarotenoid protein from A. maxima, M1 matrix protein from influenza,neuraminidase from influenza virus, E. coli fuculose aldolase; and thelike. (see, e.g., O'Shea, Science. 254: 539 (1991), Barahmand-Pour etal., Curr. Top. Microbiol. Immunol. 211: 121-128 (1996); Klemm et al.,Annu. Rev. Immunol. 16: 569-592 (1998); Klemm et al., Annu. Rev.Immunol. 16: 569-592 (1998); Ho et al., Nature. 382: 822-826 (1996); andPomeranz et al., Biochem. 37: 965 (1998)). Further examples includeresidues 325 to 410 in the bovine papillomavirus E2 protein, (Dostatni,N., et al., EMBO J 7 (1988) 3807-3816; Haugen, T., et al. EMBO J 7(1988) 4245-4253; McBride, A., et al., EMBO J 7 (1988) 533-539; McBride,A., et al., Proc Natl Acad Sci USA 86 (1989) 510-514),Type I deiodinase(D1): DFLVIYIEEAHASDGW (SEQ ID NO: 31) or ADFL-YI-EAH-DGW (SEQ ID NO:32); HIV-1 Capsid Protein: QGPKEPFRDYVDRFYKTLRA (SEQ ID NO: 33); leucinezipper dimerization motif of yeast GCN4: HMKQL D VEEL S NYHL N VARL KVGER (SEQ ID NO: 34); leucine zipper in Escherichia coli transcriptionalantiterminator protein; and BglG: GVTQLMREMLQLIKFQFSLNYQEESLSYQRLVT (SEQID NO: 35). In various specific embodiments, the third peptide domaincomprises one or more copies of a pentameric coiled coil (such asWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWM (SEQ ID NO: 25)), a trimericcoiled coil such as RLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEEL(SEQ ID NO: 26), or both. In another embodiment, the third polypeptidecomprises a flagellin protein or domain thereof, including but notlimited to:

(SEQ ID NO: 45) MAQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNV; and/or (SEQ ID NO: 46)TENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLRof Salmonella enterica flagellin.

In another aspect, the invention provides pharmaceutical compositions,comprising:

(a) the chimeric polynucleotide, the expression vector, the chimericpolypeptide, the chimeric RNA, and/or the assembly of any embodiment orcombination of embodiments of the invention ; and

(b) a pharmaceutically acceptable carrier.

As used herein, the phrase “pharmaceutically acceptable salt” refers toboth pharmaceutically acceptable acid and base addition salts andsolvates. Such pharmaceutically acceptable salts include salts of acidssuch as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic,formic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric,tartaric, maleic, hydroiodic, alkanoic such as acetic, HOOC—(CH₂)n—COOHwhere n is 0-4, and the like. Non-toxic pharmaceutical base additionsalts include salts of bases such as sodium, potassium, calcium,ammonium, and the like. Those skilled in the art will recognize a widevariety of non-toxic pharmaceutically acceptable addition salts.

The chimeric polynucleotide, the expression vector, the chimericpolypeptide, the chimeric RNA, and/or the assembly may be the soleactive agent in the composition, or the composition may further compriseone or more other agents suitable for an intended use, including but notlimited to adjuvants to stimulate the immune system generally andimprove immune responses overall. Any suitable adjuvant can be used. Theterm “adjuvant” refers to a compound or mixture that enhances the immuneresponse to an antigen. Exemplary adjuvants include, but are not limitedto, Adju-Phos, Adjumer™, albumin-heparin microparticles, Algal Glucan,Algammulin, Alum, Antigen Formulation, AS-2 adjuvant, autologousdendritic cells, autologous PBMC, Avridine™, B7-2, BAK, BAY R1005,Bupivacaine, Bupivacaine-HCl, BWZL, Calcitriol, Calcium Phosphate Gel,CCR5 peptides, CFA, Cholera holotoxin (CT) and Cholera toxin B subunit(CTB), Cholera toxin A1-subunit-Protein A D-fragment fusion protein,CpG, CRL1005, Cytokine-containing Liposomes, D-Murapalmitine, DDA, DHEA,Diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC/Alum Complex, Fowlpox,Freund's Complete Adjuvant, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP,hGM-CSF, hIL-12 (N222L), hTNF-alpha, IFA, IFN-gamma in pcDNA3, IL-12DNA, IL-12 plasmid, IL-12/GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2/lgplasmid, IL-2/lg protein, IL-4, IL-4 in pcDNA3, Imiquimod, ImmTher™,Immunoliposomes Containing Antibodies to Costimulatory Molecules,Interferon-gamma, Interleukin-1 beta, Interleukin-12, Interleukin-2,Interleukin-7, ISCOM(s)™, Iscoprep 7.0.3™, Keyhole Limpet Hemocyanin,Lipid-based Adjuvant, Liposomes, Loxoribine, LT(R192G), LT-OA or LT OralAdjuvant, LT-R192G, LTK63, LTK72, MF59, MONTANIDE ISA 51, MONTANIDE ISA720, MPL.TM., MPL-SE, MTP-PE, MTP-PE Liposomes, Murametide,Murapalmitine, NAGO, nCT native Cholera Toxin, Non-Ionic SurfactantVesicles, non-toxic mutant E112K of Cholera Toxin mCT-E112K,p-Hydroxybenzoique acid methyl ester, pCIL-10, pCIL12, pCMVmCAT1, pCMVN,Peptomer-NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic L121, PMMA,PODDS™, Poly rA: Poly rU, Polysorbate 80, Protein Cochleates, QS-21,Quadri A saponin, Quil-A, Rehydragel HPA, Rehydragel LV, RIBI, Ribilikeadjuvant system (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptide, SendaiProteoliposomes, Sendai-containing Lipid Matrices, Span 85, Specol,Squalane 1, Squalene 2, Stearyl Tyrosine, Tetanus toxoid (TT),Theramide™, Threonyl muramyl dipeptide (TMDP), Ty Particles, and WalterReed Liposomes. In one specific embodiment, the adjuvant comprises aTLR4 ligand. In another specific embodiment, the adjuvant comprisesgluco glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE).

In another embodiment, the carrier may comprise a liposome; thisembodiment may be particularly useful for polynucleotides and/or RNAembodiments of the invention.

In another aspect, the invention provides methods for treating orlimiting Toxoplasma gondii infection, comprising administering to asubject at risk of Toxoplasma gondii infection an amount effective totreat or limit the infection of the chimeric polynucleotide, theexpression vector, the chimeric polypeptide, the chimeric RNA, theassembly, or the pharmaceutical composition of any embodiment orcombination of embodiments of the invention.

As used herein, the term “subject” refers to any animal, includingmammals, preferably mice, rats, other rodents, rabbits, dogs, cats,swine, cattle, sheep, horses, or primates, and most preferably humans.

As used herein, the phrase “amount effective”, “therapeuticallyeffective amount” or “effective to treat” refers to the amount of activecompound or pharmaceutical agent that elicits the biological ormedicinal response that is being sought in a tissue, system, animal,individual or human by a researcher, veterinarian, medical doctor orother clinician.

In certain embodiments, a therapeutically effective amount can be anamount suitable for

(1) limiting development of the disease; for example, limitingdevelopment of a disease, condition or disorder in an individual who maybe predisposed to the disease, condition or disorder but does not yetexperience or display the pathology or symptomatology of the disease;

(2) inhibiting the disease; for example, inhibiting a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder; or

(3) ameliorating the disease; for example, ameliorating a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., reversing the pathology and/or symptomatology) such as decreasingthe severity of disease.

As used here, the terms “treatment” and “treating” means (i)ameliorating the referenced disease state, for example, ameliorating adisease, condition or disorder in an individual who is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., reversing or improving the pathology and/orsymptomatology) such as decreasing the severity of disease; or (ii)eliciting the referenced biological effect (e.g., reducing parasiticload or adverse effects the parasite is causing in the human itinfects).

In another embodiment, the administering comprising

(a) administering an initial dose of the chimeric polynucleotide or theexpression vector of any embodiment or combination of embodiments incombination with an adjuvant, including but not limited to GLA-SE; and

(b) administering a booster dose of the chimeric polypeptide, assemblythereof, or a pharmaceutical composition thereof. In one embodiment, thebooster composition comprises an adjuvant including but not limited toGLA-SE.

For all methods disclosed herein, the compositions are typicallyformulated as a pharmaceutical composition for administration, such asthose disclosed above, and can be administered via any suitable route,including orally, parentally, by inhalation spray, rectally, topically,or by electroporation in dosage unit formulations containingconventional pharmaceutically acceptable carriers, adjuvants, andvehicles. The term parenteral as used herein includes, subcutaneous,intravenous, intra-arterial, intramuscular, intrasternal,intratendinous, intraspinal, intracranial, intrathoracic, infusiontechniques or intraperitoneally. Dosage regimens can be adjusted toprovide the optimum desired response (e.g., a therapeutic orprophylactic response). A suitable dosage range may, for instance, be0.1 ug/kg-100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight. Thecompositions can be delivered in a single bolus, or may be administeredmore than once (e.g., 2, 3, 4, 5, or more times) as determined by anattending physician. When adjuvant is used dosage regimens can beadjusted to lower the amount of targeted antigen used to provide theoptimum desired response.

Example 1 Adjuvanted Multi-Epitope Vaccines Protect HLA-A*1101Transgenic Mice Against Toxoplasma Gondii

We created and tested multi-epitope DNA or protein vaccines with TLR4ligand emulsion adjuvant (gluco glucopyranosyl lipid adjuvant in astable emulsion (GLA-SE)) for their ability to protect againstToxoplasma gondii in HLA transgenic mice. Our constructs each includedfive of down selected CD8⁺ T cell eliciting epitopes, a universal CD4⁺helper T lymphocyte epitope (PADRE), a secretory signal, all arranged tomaximize MHC Class I presentation. Their capacity to elicit immune andprotective responses was studied using immunization of HLA-A*1101transgenic mice. These multi-epitope vaccines increased memory CD8⁺Tcells that produced IFN-γ and protected mice against parasite burdenwhen challenged with T. gondii. Endocytosis of emulsion-trapped proteinand cross presentation of the antigens may account for theimmunogenicity of our adjuvanted protein. This work demonstrates a noveladjuvanted platform assembly of peptides resulting in cross presentationof CD8⁺ T cell eliciting epitopes in a vaccine that preventstoxoplasmosis.

Results Identification of New Candidate T. Gondii SpecificHLA-A*1101-Restricted Epitopes

Five peptide epitopes that show high affinity binding to HLA-A*1101molecules, derived from SAG1, SRS52A, SAG2C, GRA6 and GRA5 haverepresentative affinities for HLA-A*1101 molecules, a haplotype thatcovered 16-30% population in China; 7-16% in Europe and North America;1.5-10% in South America.

To determine which of these peptides would be recognized in the contextof Toxoplasma infection, peripheral blood mononuclear cells (PBMC) fromT. gondii-seropositive HLA-A03 individuals were tested for response tothese peptides in pools or individually by using an IFN-γ ELISpot assay.Candidate peptides were considered immunogenic if they inducedIFN-γ-secreting spot formation that was significant compared to anirrelevant HLA-A*1101-restricted peptide. As shown in FIG. 1, there werefive peptide pools which stimulated significant response by PBMC derivedfrom Toxoplasma seropositive HLA-A03 individuals. These were: one fromSAG1₂₂₄₋₂₃₂ (KSFKDILPK (SEQ ID NO: 16)), SAG2C₁₃₋₂₁ (STFWPCLLR (SEQ IDNO: 14)), GRA5₈₋₉₈ (AVVSLLRLLK (SEQ ID NO: 17)), SRS52A₂₅₀₋₂₅₈(SSAYVFSVK (SEQ ID NO: 15)), and GRA6₁₆₄₋₁₇₂ (AMLTAFFLR (SEQ ID NO:18)).

HLA-A*1101-Transgenic Mice as A Model to Assess Cellular Immunogenicityof 5 Identified HLA-A*1101-Restricted CD8+ T Cell Epitope Peptides

To address the HLA-A11-specific genetic restriction of the 5 HLA-A*1101epitopes identified to prime for IFN-γ responses, HLA-A*1101-transgenicand C57BL/6 wild-type control mice were immunized subcutaneously withpeptides alone or mixed with GLA-SE. As shown in Table 1, robust IFN-γresponses from splenocytes were observed following immunization by apool of peptides (5 HLA-A*1101 peptides+PADRE) mixed with GLA-SE inHLA-A*1101-transgenic mice compared with C57BL/6 control mice.Immunization of HLA-A*1101-transgenic mice with GLA-SE alone did notelicit IFN-γ when splenocytes were tested with or without peptides (i.e.background levels).

Vaccination with Peptide Pools, PADRE and GLA-SE Adjuvant Protects Miceand Increase Memory Against Type II Parasite Challenge

HLA-A*1101-transgenic mice were immunized with peptide pools combinedwith GLA-SE adjuvant and PADRE three times at intervals of two weeks.PBS was used as control. Five weeks after the last immunization, micewere challenged with type II parasites. Differences in brain cystnumbers between control and immunized mice were significant (p<0.013) asshown in FIG. 2A-B, with the immunized mice having a reduced cystburden. Spleens from unchallenged immunized and control mice were testedfor the ability of the immunization to induce CD8⁺ T cell memoryresponse. As shown in FIG. 2C-D, there is an increase of memory CD8⁺ Tcells in the immunized group.

LO and AZ Multi-Epitope Polypeptide Immunogenicity In Vitro

The CD8⁺ T cell-epitopes identified are intended to form the basis ofToxoplasma vaccine for persons with the HLA-A*1101 supertype. Weexpressed and purified from E. coli a protein composed of the fiveepitopes linked in a sequence with the universal CD4⁺ T cell epitope,PADRE (AKFVAAWTLKAAA (SEQ ID NO: 19)) and the murine Igκ-chain signalsequence for targeting protein to secretory pathway at the N-terminus.The epitopes were linked together with N/K alanines or GPGPG (SEQ ID NO:23)as linker, named as LO and AZ, respectively (FIG. 3A-B). Bothproteins were purified via Ni-NTA affinity column and the molecularweight was verified by SDS-PAGE analysis (FIG. 3C). Immunogenicity ofthe two proteins compared to the pool of the 5 individual A11-restrictedCD8⁺ T cell epitope peptides was tested in vitro. Briefly, PBMCs from T.gondii seropositive HLA-A03 supertype humans were tested for theirability to generate IFN-γ in response to the stimulation with either LOor AZ protein or a pool of the peptides for 2 days to allow time forprocessing the proteins and presentation thereof to MHC class I. Thedata in FIG. 4A-C demonstrate IFN-γ secretion was significantly enhancedby stimulation with either LO or AZ multi-epitope polypeptide comparedwith a pool of the individual 5 epitope peptides (P<0.03).

Immunization with LO and AZ Multi-Epitope Polypeptides with GLA-SEAdjuvant Confers a Potent Protection in HLA-A*1101 Transgenic MiceAgainst T. gondii.

HLA-A*1101 transgenic mice were immunized with a combination of LO andAZ multi-epitope polypeptides with GLA-SE or ALUM adjuvant. As acontrol, mice were immunized with adjuvant alone or PBS. Mice were thenchallenged 2 weeks after the last immunization with type II strains ofT. gondii. As shown in FIG. 5, 80% of mice immunized with LO and AZmulti-epitope polypeptides emulsified in GLA-SE adjuvant survivedparasite challenge. In contrast, only 30% of mice immunized with theALUM adjuvant plus polypeptides survived parasite challenge (P<0.04). Asa control, neither mice immunized with the adjuvant alone nor thoseimmunized with PBS increase their survival after challenge (FIG. 5).

Prime/Boost Strategy: LO and AZ DNA Plus Multi-Epitope PolypeptideImmunogenicity In Vivo

We next addressed whether a prime/boost strategy using DNA encoding thefive individual epitopes plus PADRE and the polypeptides would conferbetter protection against parasite challenge in HLA-A*1101 mice. Weconstructed synthetic DNAs in which the 5 poly-epitope nucleotides withthe N/K alanines or GPGPG (SEQ ID NO: 23) linkers plus PADRE were clonedin EcoRI and BamHI sites of the vaccine vector pMB75.6 (FIG. 6A). DuringDNA vaccinations, mice were immunized intramuscularly (i.m.) two timesat 2 weeks interval with 100 μg of LO or AZ DNA vectors followed byanother two injections of 50 μg of the polypeptides at 2 weeks interval.They were challenged with 2,000 ME49 (Fluc) 2 weeks after the lastimmunization. Brains from these mice were imaged 21 days after thechallenge using a Xenogen in vivo imaging system to assess parasiteburden in the brain. As shown in FIG. 6B-C, the numbers of luciferaseexpressing parasites in HLA-A*1101 mice immunized with LO or AZ DNA plusmulti-epitope polypeptide or polypeptides alone were significantlyreduced compared to the mice immunized with control empty vector or PBS(P<0.02). This correlates with the reduction of the number of cysts perbrain (FIG. 6D). We then analyzed the effect of LO or AZ DNA plusmulti-epitope polypeptide on IFN-γ expression in vitro. Briefly, micewere immunized two times at 2 weeks interval with LO or AZ DNA followedby another two injections of either LO polypeptide, AZ polypeptide orovalbumin peptide as control. Negative controls mice were vaccinatedtwice with empty vector followed by saline. Two weeks after the lastimmunization, mice were sacrificed and splenocytes were harvested forimmune responses analysis. As shown in FIG. 7A, considerable amount ofIFN-γ expressing CD8⁺ cells were observed following immunization with AZDNA or LO DNA compared with mice immunized with the empty vector. Aneven more robust response was achieved when mice immunized with thevectors expressing the polypeptides were stimulated in vitro by thecorresponding AZ or LO protein, although the difference was onlysignificant in the LO treated mice. In contrast, there was no increasein the amount of CD8+ IFN-γ producing cells observed when the mice wereimmunized with AZ or LO DNA followed by a challenge with a non-relevantprotein, ovalbumin.

LO DNA Plus Multi-Epitope LO Polypeptide are Protective AgainstToxoplasma Challenge in HLA-A*1101 Transgenic Mice

As shown in FIG. 7B, a majority, 7 of 8 (87%) HLA-A*1101 mice immunizedwith LO DNA plus LO polypeptide emulsified in GLA-SE adjuvant survivedparasite challenge. In contrast, only 1 of 8 (12%) unimmunized mice orimmunized with empty vector survived parasite challenge.

LO DNA Plus Multi-Epitope Polypeptide Increases Memory CD8⁺ T CellResponse

We then analyzed the effect of LO DNA plus multi-epitope polypeptide onthe T gondii-specific CD8⁺ T cell memory response. This was performed byquantifying the levels of memory T cells in the spleen from HLA-A*1101mice at 35 days after the last immunization. As shown in FIG. 7C-D, CD8⁺memory T cells were significantly increased in mice immunized witheither LO protein alone or LODNA plus LO protein compared with miceimmunized with the empty vector or PBS.

Discussion

There is a need for improved vaccination and delivery approaches toinduce cellular immune responses against T. gondii. Herein, we present anovel way to present immunogenic peptide epitopes to a host's immunesystem based on the assembly of five protective CD8⁺ T cell epitope forHLA-A*1101-restricted supertypes. These epitopes were constructed withthe universal CD4⁺ T cell epitope, PADRE linked with N/KAAA (SEQ ID NO:21 and 22) or GPGPG (SEQ ID NO: 23) spacers and a secretory signal.These vaccine design features were incorporated to maximize proteasomeprocessing and, subsequently, epitope and vaccine immunogenicity.

Herein, we examined the immunogenicity of a multi-epitope protein andsynthetic consensus DNA, clinically approved mammalian expression vectorencoding five T. gondii specific HLA-A*1101-restricted epitopes. The DNAplasmid vaccine is encoding the five CD8⁺ T cell epitopes restricted byHLA-A*1101 supertype alleles and the universal HTL epitope, PADRE. TheseDNA constructs were optimized using codon optimization, leader sequenceaddition, plasmid production at high concentration and the DNA wasdelivered by electroporation. Immunization of HLA-A*1101 mice withrecombinant multi-epitope formulated with a Toll-like receptor 4 ligand(TLR4)-containing adjuvant (gluco glucopyranosyl lipid adjuvant in astable emulsion [GLA-SE]) induced antigen-specific IFN-γ-producing CD8⁺T cells in their spleens, increase memory CD8⁺ T cells population, andconferred a potent protection against T. gondii challenge. The adjuvantALUM formulated to the recombinant multi-epitope was less effective inconferring protection as when compared to the vaccine preparationscontaining GLA-SE. The present study also demonstrated that DNA primefollowed by a multi-epitope protein-GLA-SE boost is more protective thaneither of the DNA prime followed by ovalbumin (unrelated protein) boostor the recombinant multi-epitope alone.

Our present study showed that protein prime and boost with GLA-SE wasalso quite effective. The ex vivo stimulation of spleen cells fromHLA-A*1101 mice and PBMC from HLA-A03 seropositive individuals showedCD8⁺ T cells were more responsive to the composite polypeptide than tothe pooled or single constituent peptides. Processing and presentationof AZ and LO poly-epitope polypeptide in human cells occur with highefficiency in LO poly-epitope-stimulated cells. These cells demonstratestronger responses with N/K alanines linker compared to GPGPG (SEQ IDNO: 23).

The in vitro and in vivo murine work in the present study indicates thatthe GLA-SE nano-emulsion must provide protein for cross presentation byantigen presenting cells as shown schematically in FIG. 8. This is anovel and important finding with broad implications for vaccinedevelopment when induction of immune responses with protective CD8⁺T-cells are critical.

In summary, our study shows a composite protein, with a secretorysignal, five CD8⁺ MHC class I epitopes from T. gondii, and PADRE, can beassembled and elicits protective CD8⁺ T-cells responses. UsingHLA-A*1101 transgenic mice, we demonstrate the specificity of 5HLA-A*1101 restricted epitopes to prime and boost an IFN-γ response. Inaddition, the recombinant multi-epitope polypeptide emulsified in GLA-SEadjuvant confers more protection and increases memory CD8⁺ T cellresponse against T. gondii in HLA-A*1101 transgenic mice. It is likelythat the GLA-SE emulsion encloses the protein and presents an optimumconfiguration decorated with the TLR4 ligand GLA that induces a powerfulCD8⁺ and CD4⁺ T cell immune response (FIG. 8). In addition, DNA encodingthe multi-epitope delivered by electroporation and followed by proteinboosts is useful for the induction of a strong immune response againstT. gondii. Thus, our data provide important support suggesting thatenhanced electroporation-delivered DNA prime-protein boost, and proteinprime protein boost which would be considerably better tolerated, areuseful strategies for delivery of a multi-epitope anti-parasite vaccine.

Methods

Bioinformatic Predictions and MHC-Peptide Binding Assays

Protein sequences derived from SAG1, SRS52A, SAG2C, GRA6, and GRA5 wereanalyzed for CD8⁺ T cell epitopes based on predicted binding affinity toHLA-A*1101 molecules using algorithms available at the Immune Epitopedatabase (IEDB). Quantitative assays to measure binding of peptides toHLA class I molecules are based on inhibition of binding of radiolabeledstandard peptide. Assays were performed as described previously (Sidney,J., et al., Immunome Res, 2008. 4: p. 2). [ ]. Concentration of peptideyielding 50% inhibition of binding of radiolabeled probe peptide (IC₅₀)was calculated. Under the conditions utilized, were where [label]<[MHC]and IC50, [MHC], the measured IC50 values are reasonable approximationsof the true Kd values.

Human PBMC and ELISpot Assay

PBMC were obtained from individuals seropositive to T. gondii, and theirHLA haplotype was determined. These cells were processed andcryopreserved as described previously (Tan et al., Vaccine28(23):3977-89 (Year? Was not listed in references). ELISpot assays withhuman PBMCs used anti-human IFN-γ mAb (1-D1K) with biotinylatedanti-human IFN-γ mAb (7B6-1) with 2×10⁵ PBMCs per well. All antibodiesand reagents used for ELISpot assays were from Mabtech (Cincinnati,Ohio). The PBMC were plated in at least 3 replicate wells for eachcondition. Results were expressed as number of spot forming cells (SFCs)per 10⁶ PBMCs.

Epitope Peptides

KSFKDILPK (SEQ ID NO: 16) (SAG1₂₂₄₋₃₂), STFWPCLLR (SEQ ID NO:)(SAG2C₁₃₋₂₁), AVVSLLRLLK (SEQ ID NO: 17) (GRA5₈₉₋₉₈), SSAYVFSVK (SEQ IDNO: 15) (SRS52A₂₅₀₋₂₅₈), AMLTAFFLR (SEQ ID NO: 18) (GRA6₁₆₄₋₁₇₂) andPADRE-derived universal CD4 helper epitope (AKFVAAWTLKAAA) (SEQ ID NO:19) were used in the vaccine constructs. GLA-SE adjuvant (TLR4 agonist),synthesized by the Infectious Diseases Research Institute (Seattle,Wash.) was used as a stable oil-in-water emulsion with specifiedepitopes during immunization.

Multi-Epitope DNA Vaccine Design

To maximize epitope immunogenicity in vivo, the peptides encodingminigene included starting codon ATG and the mouse Ig k signal sequenceat the 5′ end of the construct, and spacer sequences N/AAA (SEQ ID NO:21) (LO construct) and GPGPG (SEQ ID NO: 23) (AZ construct) residuesflanking the C-terminus of all epitopes (FIGS. 3A-C). Whereas the formerfacilitates processing of the CTL epitopes in the ER, the latter favorsproper proteasomal processing and prevents the formation of junctionalHLA epitopes. The order of the CTL and HTL epitopes in the minigene andtype of spacer sequences that favor proper proteasomal cleavage weredetermined by a customized computer software program (Epimmune) thatidentifies the most favorable sequence for epitope processing andsimultaneously minimizes the creation of new junctional HLA-A11determinants.

Purification of Multi-Epitope Protein Vaccine

LO and AZ DNA were PCR-amplified and cloned in the expression vectorpET-22 (Novagen). The multi-epitope protein was expressed in theEscherichia coli BL21-CodonPlus strain (Stratagene). Expression cloneswere grown at 37° C. in Luria broth medium containing 50 μg/ul kanamycinand 34 μg/ul chloramphenicol. A 1-liter culture of E. coli was grown toan A₆₀₀ of 0.6, and protein expression was induced by addition ofisopropyl-β-D-thiogalactopyranoside (1 mM final concentration).Recombinant protein was extracted under native conditions by using theBugBuster protein extraction reagent (Novagen, 6 ml/g of cell pellet)containing a protease inhibitor mix (Roche Diagnostics) and 10 μg/mllysozyme. All purification steps were performed under 8 M ureadenaturing conditions. The His-tagged polypeptides were purified byusing nickel-affinity chromatography and followed by Q-Sepharose, whichused to capture the endotoxin. The eluate, which contains 8 M urea, wasdialyzed against a buffer containing 5 mM Hepes-KOH (pH 7.8) and 0.5 mMDTT. The purity of the protein was determined by SDS-PAGE, and theprotein concentration was measured by the method of Bradford using BSAas a standard. Using E-TOXATE Kits (Sigma-Aldrich, USA), endotoxinconcentration in these proteins is <25 EU/ug of protein.

Plasmid and Gene Cloning of Multi-Epitopes DNA Vaccine Construct

The clinically approved mammalian expression vector pMB75.6 (from InovioPharmaceuticals, Blue Bell, Pa.) was used as a DNA vaccine vector.Briefly, the plasmid contains elements essential for expression inmammalian cells: a cytomegalovirus (CMV) promoter, intron, and gene ofinterest followed by the simian virus 40 (SV40) polyadenylation signal.

The oligonucleotides of 5 individual T. gondii peptides shown in Table 1plus PADRE-derived universal CD4 helper epitope were linked withdifferent spacers and synthesized using 9 overlapping 50-nucleotideoligonucleotides. LO and AZ were first assembled and amplified as threesmall fragments that were subsequently used as templates to amplify thewhole gene. The full-length constructs were cloned into the vaccinevector pMB75.6 using EcoRI/BamHI restriction sites. Neither the pMB75.6vector backbone nor the epitope-encoding region shares significanthomology with known human genomic sequences. All recombinant plasmidswere propagated in Escherichia coli TOP10 and confirmed by restrictionanalysis and PCR sequencing. Large-scale plasmid DNA was prepared usingthe endotoxin-free Mega kit according to the manufacturer's instructions(Qiagen, Hilden, Germany), and the DNA concentrations were determined byA260/A280 absorption measurements. Plasmid DNA was dissolved in sterileendotoxin-free PBS and stored at −20° C. until use.

Mice

HLA-A*1101/K^(b) transgenic mice were produced at Pharmexa-Epimmune (SanDiego, Calif.), embryo-rederived at Taconic and JAX laboratories andbred at the University of Chicago. These HLA-A*1101/K^(b) transgenicmice express a chimeric gene consisting of the 1^(st) and 2^(nd) domainsof HLA-A*1101 and the 3^(rd) domain of H-2K^(b), and were created on aC57BL/6 background. Mice were maintained in SPF conditions throughout.All studies were conducted with the approval of the Institutional AnimalCare and Use Committee at the University of Chicago.

Immunizations of Mice and Challenge

To evaluate multi-epitope protein immunogenicity, HLA-A*1101 transgenicmice were inoculated subcutaneously (s.c.) at the base of the tail usinga 30-gauge needle with 50 μg LO or AZ recombinant protein emulsified in20 μg of GLA-SE (TLR4 agonist) three times at two weeks intervals. Forimmunization by DNA, mice were inoculated by injection of 50 ul of PBScontaining 100 μg of DNA into each quadriceps muscle using a G26 gaugeneedle at weeks 0, 2 and 4. In a bid to enhance delivery of DNA, we usedan electroporation device. Briefly, following injection of DNA, thesurface dermal device was applied to the site of injection. The arraywas “wiggled” at the injection site to ensure good contact andelectro-transfer achieved through pulse generation from the ELGEN 1000(Inovio Pharm., San Diego) pulse generator. The parameters used werethree 15 V pulses of 100 ms duration. Negative control mice werevaccinated with 100 μg empty vector or 50 μl saline. For challengestudies, immunized mice were challenged intraperitoneally (i.p.) 14 dayspost-immunization using 10,000 T. gondii ME49-Fluc (Type II) parasitesthat express firefly luciferase.

ELISpot Assay to Determine Immune Responses with Murine Splenocytes

Mice were euthanized 14 days after immunization. Spleens were harvested,pressed through a 70 μm screen to form a single-cell suspension, anderythrocytes were lysed with AKC lysis buffer (160 mM NH₄Cl, 10 mMKHCO₃, 100 mM EDTA). Splenocytes were washed twice with Hank's BalancedSalt Solution (HBSS) and resuspended in complete RPMI medium (RPMI-1640supplemented with 2 mM L-GlutaMax (Life technologies). Murine ELISPOTassays were performed using anti-mouse IFN-γ mAb (AN18) and thebiotinylated anti-mouse IFN-γ mAb (R4-6A2) and 2.5-5×10⁵ splenocyteswere plated per well. All antibodies and reagents used for the ELISPOTassay were obtained from Mabtech (Cincinnati, Ohio). Cells were platedin at least 3 replicate wells for each condition. Results were expressedas the number of spot forming cells (SFCs) per 10⁶ murine splenocytes.

In Vivo Bioluminescence Imaging for Determining Outcomes of Challengewith Type II Parasites

Mice infected with 2,000 T. gondii ME49-Fluc (Type II) tachyzoites wereimaged 21 days post-challenge using the in vivo imaging system (IVIS;Xenogen, Alameda, Calif.). Mice were injected retroorbitally with 200 μl(15.4 mg/ml) of D-luciferin, anesthetized in an O₂-rich inductionchamber with 2% isoflurane, and imaged after 12 minutes. Photonicemissions were assessed using Living image® 2.20.1 software (Xenogen).Data are presented as pseudocolor representations of light intensity andmean photons/region of interest (ROI). All mouse experiments wererepeated at least twice.

Enumeration of Cysts in Mouse Brains Following Type II ParasiteChallenge

Mice were euthanized at 21 days after infection with 10,000 ofMe49-Fluc, and brains were collected, homogenized with 1 ml of saline(0.85% NaCl). Tissue cysts were counted microscopically in 50 μl of thehomogenate, and the count was multiplied by 20 to obtain the number oftissue cysts per brain. This number was confirmed by staining braincysts with fluorescein-labeled Dolichos biflorus agglutinin (VectorLaboratories) and quantitation using fluorescence microscopy.

Flow Cytometry.

Splenocytes were manually processed using 70 μm filters in DMEM mediasupplemented with 5% FCS and red blood cells were lysed with ACK lysisbuffer. Cells were stained with CD3 APC (145-2C11), CD4 PE (GK1.5), CD8PerCP (53-6.7), and CD44 AF780 (IM7), CD45RB FITC (C363.16A). Allantibodies were purchased from eBioscience (San Diego, Calif.). Memory Tcells were defined as CD44^(hi)CD45RB^(lo). All flow cytometry data wascollected on LSRII flow cytometer (BD Biosciences, San Jose, Calif.) andanalyzed using FlowJo software 10.0 (Tree Star, Ashland, Oreg.).

Statistical Analyses

Data for each assay were compared by One-way ANOVA or a Student t testusing GraphPad Prism 5 software (GraphPad Software, San Diego, Calif.).Differences between the groups were identified by ANOVA and multiplecomparison procedures, as we previously described [6]. Data areexpressed as the means±SD. Results were considered to be statisticallysignificant at p<0.05.

Study Approval

Experiments and handling of mice were conducted under federal, state,and local guidelines under an IACUC protocol and with approval from theUniversity of Chicago IACUC. Institutional Review Board (IRB) approvalwas obtained at the University of Chicago for this study. This studyalso is in compliance with all Health Insurance Portability andAccountability Act of 1996 (HIPAA) regulations.

Example 2 Novel Protein Nanovaccine Confers Robust Immunity AgainstToxoplasma

We designed and produced a self-assembling protein nanoparticle (SAPN)containing five CD8⁺ HLA-A03-11 supertypes restricted epitopes fromantigens expressed during Toxoplasma's lifecycle, PADRE which is a CD4⁺T cell, universal epitope, and flagellin as scaffold and TLR5 agonist.These epitopes were separated by N/KAAA (SEQ ID NO: 21 & 22) spacers andoptimized for proteasomal cleavage. SAPN-GLA-SE was evaluated for itsefficacy in inducing IFN-γ and protection against T. gondii in mice withan HLA-A*1101 transgene. In our data, immunization with SAPN adjuvantedwith TLR4 ligand-emulsion (GLA-SE), activated CD8⁺ T cells to produceIFN-γ. SAPN-GLA-SE also protected against subsequent challenge with typeII parasites in mice with an HLA-A*1101 transgene. Hence, combining CD8⁺T cell-eliciting peptides and PADRE into a multi-epitope within a singleself-assembling protein, administered with adjuvant GLA-SE, leads toefficient presentation by MHC Class I and II. Furthermore, these resultssuggest that activation of TLR4 and TLR5 could be useful for developmentof T cell vaccines to prevent toxoplasmosis in humans.

In the present study, five epitopes from the surface antigen (SAG1), thedense granule proteins (GRA3 and GRA6), the surface antigen-1-relatedsequences (SRS52A), which bind to HLA-A11-01 were evaluated for theirefficacy as a SAPN-vaccine in HLA-A11-01 transgenic mice. In theseconstructs, the CD8⁺ HLA-A03-11 supertypes restricted epitopes werelinked by N/KAAA (SEQ ID NO: 21 and 22) spacers and conjugated withPADRE, a universal CD4⁺ helper T lymphocyte epitope. PADRE bindspromiscuously to MHC class II variants, augments effector functions ofCD8+ T cells by producing IL2, which augments induction of CD4⁺ T helpercells (Brown, et al., Journal of immunology 145, 3438-3441 (1990);Gazzinelli, et al. Journal of immunology 146, 286-292 (1991)). Epitopeseliciting both CD4⁺ and CD8⁺ T cells are important components in theformulation of successful vaccines by driving a protective response. Ourdata herein show that incorporating this peptide into the SAPN proteinconstructs and delivering this in TLR4 ligand emulsion adjuvant(GLA-SE), resulted in activation of CD8⁺ T cells. This led these cellsto produce IFN-γ. They thereby protected against subsequent challengewith type II parasites given as a high inoculum. Thus, our workhighlights the potential for the use of SAPN as a platform for thedelivery of CD8⁺ and CD4⁺-restricted epitopes, in adjuvant formulationto protect against toxoplasmosis.

Results

Preparation and Characterization of CD8-SAPN and Empty-SAPN. The SAPNconstructs were expressed, purified and folded to form nanoparticles.The protein has a relative molecular weight of about 48 kDa on aSDS-PAGE (data not shown). Transmission electron microscopy (not shown)showed a relatively uniform distribution of non-aggregated nanoparticlesof ˜30 nm in diameter.

In vivo Immunogenicity of CD8-SAPNs. Mice with HLA-A*1101-transgeneswere immunized. Immunogen was CD8-SAPNs combined with GLA-SE adjuvant.Immunizations were given three times intramuscularly at two weekintervals. Empty-SAPNs plus GLA-SE adjuvant and PBS were used ascontrol. Immunogenicity of the CD8-SAPNs plus adjuvant compared to theEmpty-SAPN plus adjuvant were compared in immunized mice with HLA-A*1101transgenes as described. Spleen cells were obtained from immunizedHLA-A*1101 transgenic mice two weeks after final immunization. IFN-γwhen cultured with the pool of peptides. FIG. 9 shows IFN-γ secretion ishigh in mice immunized with CD8-SAPN plus GLA-SE under stimulationeither of PADRE or GRA6 peptide. The other peptides also elicited IFN-γresponse. The best response, was with the polyepitopes. FIG. 10a-bindicate IFN-γ secretion was significantly enhanced by immunization withHLA-A*1101-restricted peptide epitopes eliciting CD8⁺ T cells and notwith Empty-SAPN or PBS when cells are stimulated withHLA-A*1101-restricted peptide epitopes that elicit CD8⁺ T cells.Significantly more IFN-γ secretion was observed when cells arestimulated with pool of peptides plus PADRE. Thus, the association ofCD8⁺ T cell and CD4⁺ T cell restricted peptides contributes to IFN-γproduction in mice with HLA-A*1101 transgenes.

In vitro TLR5 Stimulation. SeaPorter™ TLR5 cell-line was exposed tovarying concentrations. The concentrations used in ng/ml were 0.01, 0.1,1, 10, 100, 1000 of Empty-SAPN that don't contain the CD8⁺ epitopes butstill have flagellin, CD8-SAPN containing the polypeptide with the fiverestricted CD8⁺, epitopes, and recombinant flagellin (as control). Thelevel of TLR5 stimulation was determined by the level of SEAPexpression. Fold increase in SEAP expression for each protein sampleover non-treated controls. As shown in FIG. 11A-C, TLR5 activity wassignificantly enhanced in the Empty-SAPN and the CD8-SAPN and not in thecontrol polypeptide. Surprisingly, flagellin in Empty-SAPN particleshave higher TLR5 activity than recombinant flagellin alone.

SAPN with GLA-SE Adjuvant Confer Robust Protection Against T. gondii inmice with HLA-A*1101 Transgenes. We immunized mice by administeringeither CD8-SAPN with GLA-SE adjuvant or Empty-SAPN with GLA-SE adjuvant,adjuvants alone, PADRE alone, saline and then challenged two weeks afterthe last immunization with type II strains of T. gondii. Brains fromthese mice were imaged. This xenogen camera imaging took place 21 dayschallenge with 2,000 Me49-Fluc. This was in vivo imaging system byXenogen. FIG. 12A-B luminescence in mice that were immunized withCD8-SAPN plus GLA-SE were significantly lower when they were comparedwith mice immunized with control Empty-SAPN plus GLA-SE, GLA-SE alone,PADRE alone, or PBS. This correlates with the reduction of the number ofcysts per brain in CD8-SAPN plus GLA-SE adjuvant mice (FIG. 12c ).

Discussion

Our capability to control the ability of peptides and proteins toself-assemble into particles well-defined size a shape allows us todesign mechanically and chemically stable. Since the SAPNs resemblevirus capsids they combine strong immunogenic effect of live attenuatedvaccines with high specificity in eliciting immune responses ofprotein-based vaccines. It is apparent that the SAPN have a greatpotential to serve as a platform for vaccines beyond their ability torepetitively present antigens. In contrast to live attenuated vaccines,SAPN-derived vaccines pose no risk of infection. They are very versatileand flexible in their design leading to better biophysical andimmunological properties. Furthermore, of bacterial protein expression,purification, and self-assembly into nanoparticles reduces timelarge-scale production.

Here we used the SAPNs to present immunogenic peptide epitopes to ahost's immune system based on the assembly of five protective CD8⁺ CTLHLA-A03-11 restricted supertypes with PADRE in a SAPN. All epitopes wereflanked at the carboxy-terminus by N/KAAA (SEQ ID NO: 21 and 22)spacers, which promote optimally immunogenic processing. Our data showpotent immunogenicity (high IFN-γ secretion) when splenocytes werestimulated in vivo by these peptides. We found flagellin in a SAPN madeprotection against influenza much more robust. In the TLR5 activityassay the SAPN shows good stimulation, however, the activity is reducedcompared to the Empty-SAPN. There might be some interference withTLR5-binding and the presentation of the CD8⁺ restricted epitopes as theCD8⁺ epitopes string was engineered into the flagellin molecule toreplace the D2 and D3 flagellin domains.

In the work with this new SAPN-design herein, the flagellin moleculeitself serves as a scaffold for the T. gondii peptides, while theflagellin molecule itself is an integral part of the SAPN scaffold, withor without the A11 CD8+ epitopes. This SAPN scaffold lacking the CD8+epitopes conferred only a small amount of protection compared with thescaffold with the inclusion of the A11 peptides. It is not possible tocreate a relevant separate control without flagellin because in thisscaffold the HLA A11 binding peptides are intercalated into theflagellin molecule itself. As can be seen in FIG. 1, these A11 peptidesare an integral part of the flagellin molecule within the SAPNs. Theinclusion of flagellin also serves as a potential adjuvant.

In summary, our study shows a self-assembling protein nanoparticle, withfive CD8⁺ MHC class I epitopes from T. gondii and PADRE, can beassembled. Using HLA-A*1101 transgenic mice, we demonstrate that theSAPN emulsified in GLA-SE adjuvant elicits a protective MHC class Iresponse. Thus, our work indicates a novel improved assembly of peptidesfor cross presentation of CD8⁺ T cell eliciting epitopes in vaccines toprevent toxoplasmosis.

Materials and Methods

-   Peptides. KSFKDILPK (SEQ ID NO: 16) (SAG1₂₂₄₋₂₃₂), STFWPCLLR (SEQ ID    NO: 14) (SAG2C₁₃₋₂₁), AVVSLLRLLK (SEQ ID NO: 17) (GRA5₈₉₋₉₈),    SSAYVFSVK (SEQ ID NO: 15) (SRS52A₂₅₀₋₂₅₈), AMLTAFFLR (SEQ ID NO: 18)    (GRA6₁₆₄₋₁₇₂)²³ and PADRE, a universal CD4⁺ helper epitope    (AKFVAAWTLKAAA) (SEQ ID NO: 19) were used in the vaccine constructs.    Infectious Diseases Research Institute (Seattle, Wash.) synthesized    the TLR 4 agonist adjuvant called GLA-SE. This was prepared and used    as a stable oil-in-water emulsion.-   Molecular Biology. The methods using DNA coding for the nanoparticle    constructs were similar to those described in our earlier work¹⁸.    Briefly, they were prepared using standard molecular biology    procedures. Specifically, plasmids containing the DNA coding for the    protein sequence were used¹⁸. They were constructed by cloning into    restriction sites in the SAPN expression plasmid¹⁸. We used a SAPN    construct we had developed and described earlier (Babapoor, S. et    al. Influenza research and treatment 2011, 126794,    doi:10.1155/2011/126794 (2011)). Briefly, this construct is composed    of a pentameric coiled-coil tryptophane zipper. This zipper is    linked by a glycine residue to a trimeric de-novo designed leucine    zipper coiled coil. In this construct, a flagellin construct    composed of the D0 and D1 domains (residues 1-177 and 249-372) of    Salmonella enterica flagellin from the structure with pdb-code 3V47    from the RCSB protein data bank is used to extend the protein chain    at the C-terimnus¹⁸ (FIG. 1).-   The CD8⁺-peptide sequence    AVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAA W    TLKAAAKSTFWPCLLR (SEQ ID NO: 36) with the five CD8⁺ epitopes also    containing PADRE was next inserted into the D1 domain of flagellin.    This polypeptide completely replaces the D2 and D3 domains to    generate the so-called CD8-SAPN. Overall, the positive charge of    this epitope string is balanced with stretches of negative charges    at both ends of the epitope sequence. Our Empty-SAPN was generated    using the short linker KYKDGKGDDK (SEQ ID NO:38) to replace the D2    and D3 domains of flagellin.-   Protein Expression. This was performed as performed and described in    Babapoor et al: Plasmids were transformed into Escherichia coli BL21    (DE3) cells. E. coli were grown at 37° C. in Luria broth with    ampicillin. We induced expression using isopropyl    β-D-thiogalacto-pyranoside. Cells were removed from 37° C. four    hours after induction. They were harvested by centrifugation at    4,000×g. We stored cell pellet at −80° C. We then thawed the cell    pellet, keeping this on ice. We then suspended this in a lysis    buffer consisting of 9 M urea, 100 mM NaH₂PO₄, 10 mM Tris pH 8, 20    mM imidazole, and 0.2 mM Tris-2-carboxyethyl phosphine (TCEP).    Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)    was used to assess our protein expression level.-   Protein Purification. The same methodology as used in Babapoor, S.    et al. was used herein. Briefly, sonication was used to lyse cells.    Briefly, centrifuging at 30,500×g for 45 min¹⁸ was used to clear our    lysate. Then, for at least 1 hour, our cleared lysate was incubated    with Ni-NTA Agarose Beads (Qiagen, Valencia, Calif., USA). Then,    first the column was washed with lysis buffer. This was then    followed by a wash with a buffer containing 9 M urea, 500 mM    NaH₂PO₄, 10 mM Tris pH 8, 20 mM imidazole, and 0.2 mM TCEP. A pH    gradient was used to elute our protein. This pH gradient for elution    was created as follows: 9 M urea, 100 mM NaH₂PO₄, 20 mM citrate, 20    mM imidazole, and 0.2 mM TCEP. Subsequent washes were performed as    follows: at pH 6.3, 5.9, and 4.5. After the pH gradient, to further    elute the protein, we used a gradient of lysis buffer with    increasing imidazole concentration.-   Protein Refolding. We used methodology described in Babapoor, S. et    al. Specifically, for refolding, the protein was first rebuffered to    the following conditions: 9M urea, 20 mM Tris pH 8.5, 50 mM NaCl, 5%    glycerol, 2 mM EDTA. Four μl of a solution with a concentration of    1.8 mg/ml protein was added to the same buffer solution without urea    to a final concentration of 0.05 mg/ml for quick refolding of a    first screen. We used this because this quick dilution from    denaturing (urea) to native (no urea) buffer conditions triggers    refolding of protein. We then used negative stain transmission    electron microscopy at different resolutions to analyze our    solution. We then used further screens for optimal refolding    conditions. These were performed with smaller sampling sizes of the    pH and ionic strength.-   In vitro TLR5 Response Assay. Activation through TLR5 was assessed    for SAPN as described in Karch, et al. Nanomedicine,    doi:10.1016/j.nano.2016.08.030 (2016)). Testing was done using    TLR/NF-κB/SEAPorter™ Stably Transfected HEK 293 Cell Lines as    follows: All cell lines were stably co-transfected cell lines. They    express TLR5 and secreted alkaline phosphatase (SEAP) reporter gene    under transcriptional control of an NF-κB response element. Fourteen    thousand cells per well were seeded in a 96 well plate at passages    5-9. Twenty-four hours later, we removed growth media. Growth media    was replaced with DMEM high glucose (Hyclone, Logan, Utah). This    contained either a SAPN, or recombinant flagellin (Novus    Biologicals, Littleton, Colo.), 0.1, 1, 10, 100, 1000 ng/mL in    triplicate. Media alone was present in control wells. Wells were    exposed for 24 hours. Then supernatant was collected. Supernatant    was used to determine whether secreted alkaline phosphate was    present. This was determined with a Reporter Assay kit for secreted    alkaline phosphates (Novus Biologicals, Littleton, Colo.). This was    done using the manufacturer's instructions. Media only controls were    used to normalize SEAP activity. This was used to determine each    construct's EC50. Triplicate determinations were utilized for each    experimental condition.-   Mice. HLA-A*1101/K^(b) transgenic mice were created and then    bred/produced at Pharmexa-Epimmune (San Diego, Calif.). They were    then embryo-rederived at Taconic and JAX laboratories. Colonies were    then expanded and they were then maintained and produced in    isolators at the University of Chicago. These mice express a    chimeric gene called HLA-A*1101/K^(b) transgene. This chimeric gene    consists of the 1^(st) and 2^(nd) domains of HLA-A*1101 and the    3^(rd) domain of H-2K^(b). Mice were maintained in SPF conditions    throughout. All of our studies were performed with the Institutional    Animal Care and Use Committee at the University of Chicago's review,    approval, and oversight.-   Immunizations of Mice and Challenge. To assess the immunogenicity of    the SAPNs, mice with the HLA-A*1101 transgene were inoculated    intramusculary (i.m.). In this injection, 20 μg SAPN was emulsified    in the TLR4 agonist, i.e., 5 μg of GLA-SE. The immunizations were    three times at two weeks intervals. For the experiments in which    these mice were challenged, challenge was at 14 days    post-immunization. Specifically, they were challenged using 2,000    Type II (Me49-Fluc) parasites. These parasites were injected    intraperitoneally.-   ELISpot Assay to Determine Immune Responses with Murine Splenocytes.    Mice were euthanized 14 days after immunization. Spleens were    harvested as follows: Initially, they were pressed through a 70 μm    screen. This allowed for formation of a suspension of single-cells.    Erythrocytes were depleted from this suspension. AKC lysis buffer    (160 mM NH₄Cl, 10 mM KHCO₃, 100 mM EDTA) was used to deplete the    RBCs. Hank's Balanced Salt Solution (HBSS) was used to was    splenocytes twice. Then they were resuspended. The medium used to    resuspend them was RPMI-1640 supplemented with 2 mM L-GlutaMax.    Murine splenocyte ELISPOT assays were performed as described    earlier. This was done using anti-mouse IFN-γ mAb (AN18) and    biotinylated anti-mouse IFN-γ mAb (R4-6A2). In each well, 2.5-5×10⁵    splenocytes were plated. Mabtech (Cincinnati, Ohio) was the source    of all of the antibodies and all of the reagents used to perform    ELISPOT assays. A mimimum of 3 three replicate wells were used to    plate cells for each condition, as we described earlier, to measure    spot forming cells (SFCs) per 10⁶ murine splenocytes.-   Bioluminescence Imaging for Determining Outcomes of Challenge with    Type II Parasites. We imaged mice infected with 2,000 Fluc    tachyzoites of the Me49 strain of T.gondii. These mice were imaged    21 days after challenge. An in vivo imaging system (IVIS; Xenogen,    Alameda, Calif.) allowed us to visualize luciferin injected    retroorbitally interacting with luciferase in the parasites. These    mice were anesthetized. Anesthesia was performed in an O₂-rich    induction chamber with 2% isoflurane. Twelve minutes after receiving    luciferin, imaging took place. Living image® 2.20.1 software    (Xenogen) was used for assessment of photonic emissions. Pseudocolor    representations of light intensity and mean photons/region of    interest (ROI) represent parasite burden in the imaging. All these    mouse experiments were replicated a minimum of two times as in our    earlier work²³. In each group we used five mice²³.-   Enumeration of Cysts in Mouse Brains Following Type II Parasite    Challenge. Mouse brains were collected at day 21. They were    homogenized in 1 ml of saline (0.85% NaCl). 50 μl of the homogenate    was the used to count tissue cysts, microscopically. Cyst count was    then multiplied by 20. This product was then used to determine the    number of tissue cysts per brain. This number was confirmed by    staining brain cysts with fluorescein-labeled Dolichos biflorus    agglutinin (Vector Laboratories) and quantitation using fluorescence    microscopy.-   Statistical Analyses. Data were compared for each assay by ANOVA and    a Student t test. GraphPad Prism 5 software (GraphPad Software, San    Diego, Calif.) as described. Differences between the groups were    identified by ANOVA and multiple comparison procedures, as we    previously described. Means±SD are used to express data. A p    value<0.05 was considered to be statistically significant for our    results.

Example 3 Novel, Immunogenic, Self-Replicating RNA Nanoparticle,Platform Encoding Immunosense Designed Toxoplasma Peptides is A VaccineThat Protects HLA-A*1101 Transgenic Mice

We designed and produced a self-replicating RNA nanoparticle displayingpeptide epitopes that induces protective CD8⁺ and CD4⁺ T cells againstT. gondii infection. These RNA replicons are composed of VenezuelanEquine Encephalitis (VEE) alphavirus backbone, 5 CD8+ HLA-A03-1101supertype-restricted epitopes from antigens expressed during the lifecycle of Toxoplasma, and a universal CD4⁺ T cell epitope (PADRE). Allare encapsulated in lipid nanoparticles and evaluated for theirimmunogenic and protective potential against T. gondii in HLA-A*1101transgenic mice. Administered without the need for an adjuvant, theself-replicon nanoparticles elicit T cells producing IFN-γ and protectmice against parasite burden when challenged with type II T. gondiistrain. Thus, this work demonstrates that RNA replicon nanoparticles canpresent selected antigenic epitopes that elicit CD8+ T cells thatproduce interferon y and thereby act as a powerful immunostimulatoryprotective vaccine.

Materials and Methods RNA Synthesis

The DNA plasmid contains alphavirus RNA replication machinery genesencoding the self-amplifying RNA (pTK159) and our linked 5 HLA*A-1101antigenic peptides plus PADRE is synthesized by GENEWIZ. The RNAsequence is as follow:

(SEQ ID NO: 29) GAAUUUGCUGCGACGAUGGGUAUGCAAGUCCAGAUCCAGAGCCUGUUCCUGCUGCUGCUGUGGGUGCCGGGUUCACGCGGUAUGGCGGUGGUUAGCCUGCUGCGUCUGCUGAAAAACGCCAUGCUGACCGCAUUUUUCCUGCGCAAUGCGGCCGCAAAGAGUUUCAAGGAUAUCCUGCCGAAAAAGGCUGCGGCCAGCUCUGCGUAUGUCUUUUCCGUGAAAAAGGCAGCUGCGAAAUUCGUUGCUGCCUGGACCCUGAAAGCUGCCGCUAAAUCGACGUUCUGGCCGUGUCUGCUGCGU

The encoded sequence is:

(SEQ ID NO: 37) EFAATMGMQVQIQSLFLLLLWVPGSRGMAVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLR STOP.

Plasmids were amplified from E. coli and purified using Qiagen PlasmidMaxi kits (Qiagen). DNA is linearized by restriction enzyme digestion.Linearized DNA templates were transcribed into RNA and purified usingMEGA script T7 kit (Life Technologies) and purified using lithiumchloride (LiCl) procedures. The self-RNA replicating was thenencapsulated with lipids using the Vaccinia Capping system (New EnglandBioLabs) and purified by LiCl precipitation before formulation.

Mice

We used HLA-A*1101 transgenic mice that express a chimericHLA-A*1101/H2-Db MHC Class I Molecule and are on a C57BL/6×Balb/Cbackground backcrossed to study the 5 HLA-A*1101 peptides responses.They originally were produced at Pharmexa-Epimmune (San Diego, Calif.)and bred at the University of Chicago.

Protein Synthesis

5 HLA*A-1101 antigenic peptides plus PADRE were linked with the spacersequences N/AAA (SEQ ID NO: 21). To make proteins, DNA was PCR-amplifiedand cloned in the expression vector pET-22 (Novagen). The multi-epitopeprotein was expressed in the Escherichia coli BL21-CodonPlus strain(Stratagene). Proteins were purified using standard protocols.

Immunogencity of RNA Nanoparticles

To test the RNA nanoparticles, self-assembling RNA replicon encodingToxoplasma immunogenic antigens and PADRE were evaluated forimmunogenicity in mice. Purified RNA (1 μM) was injectedintramuscularly, 50 μL in each quadriceps muscle, in mice as a prime anda boost. 10 days after the boost, spleens from immunized were harvested,pressed through a 70 μm screen to form a single-cell suspension, anddepleted of erythrocytes with ACK lysis buffer (160 mM NH₄Cl, 10 mMKHCO₃, 100 μM EDTA). Splenocytes were then washed twice in PBSsupplemented with 5% FBS (Atlanta Biologicals, Flowery Branch, Ga.), andresuspended in complete RPMI medium (RPMI-1640 supplemented with 2 mML-GlutaMax (Life Technologies), 100 U/ml penicillin, 100 μg/mlstreptomycin, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, and 10%FBS) before use in the ELISpot assay. To evaluate protective effects ofRNA replicons in vivo, immunized HLA-A*1101 transgenic mice werechallenged with 10,000 type II (Me49-Fluc) parasites and parasite burdenquantitated as described previously (10).

Enzyme-Linked Immunospot (ELISpot) Assay

Multiscreen MSIPS4W10 HTS-IP filter 96-well plates (Millipore,Billerica, Mass.) were washed first with 35% ethanol for 1 min andincubated with 15 μg/ml α-mouse IFN-γ capture mAb (AN18; Mabtech,Cincinnati, Ohio) in sterile PBS overnight, and washed with sterile PBS.The plate was blocked with RPMI-1640 medium containing 10% FBS at roomtemperature for 2 hours. ˜2-5×10⁵ murine splenocytes were plated perwell and incubated with CD8+ HLA-A03-11 supertypes restricted epitopepeptides at 10 μg/ml for 48 hours at 37° C. Plates were then washed withsterile PBS and incubated with 1 ng/ml biotinylated α-mouse IFN-γdetection mAb (R4-6A2) for 2 hours at room temperature, washed with PBS,incubated with streptavidin-conjugated alkaline phosphatase at 1/1000dilution for 1 hour at room temperature. Spots were developed using5-bromo-4-chloro-3-indolyl-phosphate/p-nitro blue tetrazolium chloride(BCIP/NBT). The spots were counted using an automated ELISpot reader(CTL Immunospot).

Cyst Count in Mouse Brains Following Type II Parasite Challenge

Mice brains collected at day 21 were homogenized in saline and tissuecysts were counted in a mounted-slide using optical microscope. Thecysts are confirmed with fluorescein-labeled Dolichos biflorusagglutinin (Vector Laboratories).

Statistical Analyses

Statistical analyses for all applicable assays were performed using a2-tailed student's t-test.

Results Development of RNA Replicons as a Platform for Peptides Delivery

We produced RNA replicons containing 5 linked CD8+ HLA-A03 supertypesrestricted epitopes from highly immunogenic antigens described above andlinked with N/AAA spacer with PADRE. The self-assembling RNA derivedfrom an alphavirus genome contains the genes encoding the viral proteinsrequired for RNA replication. The structural protein genes were replacedwith our composite linked antigens eliciting protective CD8⁺ and CD4⁺ Tcells. The size and RNA encapsulation were characterized. Encapsulationof RNA was determined by dynamic light scattering (data not shown). Forthis study, we tested 3 lipid nanoparticles (LNP); empty LNPs (withoutRNA), LNP-encapsulated RNA without Toxoplasma antigens (pTK159) todetermine whether RNA encapsulation is necessary for efficient RNAdelivery and reporter gene expression, LNPs with Toxoplasma insert (pTK426)

RNA Replicons with HLA*A-1101-Restricted CD8+ T Cell Epitope PeptidesImmunogenicity In Vitro

HLA-A*1101 transgenic mice were immunized twice at intervals of threeweeks with RNA replicons. Two weeks after the last immunization, thespleens were removed from immunized mice and the ability of splenocytesto produce IFN-γ upon stimulation with peptides or full-length proteinwas analyzed. Empty RNA and pTK159 were used as control. FIG. 13Aindicates IFN-γ secretion was significantly enhanced by immunizationwith self-assembling RNA containing Toxoplasma insert and not withcontrol RNAs when splenocytes were stimulated with either pool ofHLA*A-1101-restricted CD8+ T cell epitope peptides or whole protein(FIG. 13B). These results demonstrate that HLA*A-1101-restricted CD8+ Tcell epitopes and PADRE in this nanoparticle RNA vaccine delivers helpof IFN-γ production.

Protective Immune Responses of Encapsulated Self-Assembling RNA AgainstType II Strain in Mice

HLA*A-1101 mice were immunized and challenged 2 weeks after the lastimmunization with type II (Me49 Fluc) parasites. A majority, 4 of 5(80%) mice immunized with pTK426 survived parasite challenge. Incontrast, only 2 of 5 (40%) immunized with empty RNA survived parasitechallenge. Mice immunized with composite protein were less protectedthan mice immunized with self-assembling RNA.

Discussion

Herein, we present a novel way to present Toxoplasma immunogenic peptideepitopes to a host's immune system based on a self-assembling RNAreplicon. This RNA is based on VEE alphavirus backbone. The RNA encodesfor non-structural proteins, involved in RNA replication and insert ofT. gondii peptides. The selection of peptides was based on previousstudies that show these peptides elicited immuneresponse and activated Tcell responses. All are encapsulated in lipid self-assembling RNAnanoparticles and used for vaccination. This approach showed to be safeand confered protection from type I allergy.

These RNA nanoparticles were evaluated for their efficacy in elicitingIFN-γ by HLA-A*1101 transgenic mice and for their protection againstToxoplasma challenge. This work demonstrates the feasibility of usingthis RNA platform for the delivery of potentially protective restrictedby human major histocompatibilty (HLA) class I in our model HLA-A*1101mice.

The self-amplifying RNA based on an alphavirus genome contains the genesencoding the alphavirus RNA replication machinery, and the genesencoding the parasite insert combines epitopes from T.gondii with thisnew methodology for RNA delivery. The genes encoding the antigenreplaced the genes encoding the viral structural proteins that arehighly expressed in the cytoplasm. Therefore it is important to have asystem, which is driving the expression of antigens in this location. Nolive viral subunits were generated by the self-assembling of the viralnanoparticles. After immunization, replication and amplification of RNAleading to self-assembling of RNA occurs in the cytoplasm. Therefore,delivery barriers for the epitopes are excluded with this model. Wefound that these RNA particles are highly immunogenenic compared to theempty RNA or peptide with GLA adjuvant (data not shown). However, thereis no protection against Toxoplasma type I strain (data not shown).Although the induction of interferon protection is robust, theprotection against type II T.gondii is not complete. By using anadjuvant, perhaps even something like profiling encoded in the RNA, orinclusion of other epitopes that elicit helper T cells, or addinginvasion blocking B cell epitopes, the efficacy of the RNA nanoparticlevaccine can be increased. It also will be of interest to our RNAreplicon vaccines to add protective peptide epitopes responses for threeHLA supermotifs, A02, A03 and B07 present in 90% of people and studyresponses in the supertype mice HLA-A*0201, HLA-A11*01, and HLA-B*0702.

Example 4 Protection and Harm from CD4⁺ T Cell-Eliciting Vaccines

The role of CD4⁺ T cells for the control of both acute and chronicToxoplasma infection is relatively unexplored. Previous studiesidentified a protective role of CD4⁺ T Cell-stimulating peptide, AS15 inC57BL/6 brain mice. Here, we show that a boost immunization ofHLA-A*1101 mice with AS15 administered with GLA-SE adjuvant, robustlyincrease cytokines expression (IFN-γ, TNF α, and IL6) and leads to gutpathology. The mice that survived the cytokines storm confer robustprotection against type I strain parasites. Introduction of CD8⁺ T-cellstimulating T. gondii peptides or the universal CD4³⁰ helper T cellepitope, PADRE, in a vaccine formulation with adjuvanted AS15 suppressthe necrosis and reduces IFN-γ production. Thus, these findingsdemonstrate quantitative and qualitative features of an effectiveToxoplasma-specific CD4⁺ T cell response that should be considered inthe testing of the next generation of different vaccine designs againsttoxoplasmosis.

Materials and Methods Peptides and Adjuvants

The CD4⁺ T cell-stimulating peptide AS15 (AVEIHRPVPGTAPPS (SEQ ID NO:20)), the universal human CD4⁺ T cell eliciting epitope, PADRE(AKFVAAWTLKAAA (SEQ ID NO: 19)), the CD8⁺ T cell-stimulating peptidesGRA6 (AMLTAFFLR (SEQ ID NO: 18)), SAG1 (KSFKDILPK (SEQ ID NO: 16)),SAG2C (STFWPCLLR (SEQ ID NO: 14)), SPA (SSAYVFSVK (SEQ ID NO: 15)), SPA(AVVSLLRLLK (SEQ ID NO: 17)) were synthesized by Synthetic Biomolecules,San Diego at >90% purity in lyophilized form. The TLR4 agonist was aGLA-SE mimetic that was synthesized by the Infectious Diseases ResearchInstitute (IDRI, Seattle, Wash.).

Analysis of Peptide Solubility

Peptides were dissolved in DMSO. Samples were centrifuged at 15,000 rpmat room temperature for 1 h. Supernatant concentrations were determinedby measuring absorbance at 280 nm and by HPLC. The percentage solubilityof the samples was calculated, although no detailed studies of optimalconcentrations of peptides or adjuvant were performed herein.

ELISpot Assays

In order to measure the IFN-γ production spleen, ELISpot assays wereperformed. Mice were euthanized 7 to 14 days after immunization. Spleenswere harvested, and depleted of erythrocytes with AKC lysis buffer (160mM NH4C1, 10 mM KHCO3, 100 M EDTA). Splenocytes were washed twice withHank's Balanced Salt Solution (HBSS) and suspended in complete RPMImedium (RPMI-1640 supplemented with 2 mM L-GlutaMax [Invitrogen], 100U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 50M-mercaptoethanol, and 10% FCS) before they were used in subsequent invitro assays. Peptide concentration used for stimulation of splenocyteswas 20 mg/ml. ELISpot assays with murine splenocytes were performedusing α-mouse IFN-γ mAb (AN18) and biotinylated α-mouse IFN-γ mAb(R4-6A2) as the cytokine-specific capture antibodies. Antibodies weremonoclonal antibodies. 5×10⁵ splenocytes were plated per well.

Mice

Female HLA-A*1101 transgenic mice express a chimeric HLA-B07/H2-Db MHCClass I molecule, are on a C57BL/6×Balb/c background backcrossed throughmany generations and have been previously described [8, 12], which wereproduced at Pharmexa-Epimmune (San Diego, Calif.) and bred at theUniversity of Chicago. All studies were conducted with the approval ofthe Institutional Animal Care and Use Committee at the University ofChicago.

Challenge Experiments

We infected the HLA-A11 mice with a type II strain (ME49) of T. gondii,which is most frequently associated with human toxoplasmosis. TheHLA-A11 mice were inoculated subcutaneously at the base of the tailusing a 30-gauge needle with the peptides described earlier. Controlmice were injected with PBS. The mice were challenged intraperitoniallywith 2000 ME49 tachyzoites 10-14 days after the last immunization.

In Vivo Bioluminescence Imaging for Determining Outcomes of Challengewith Type II Parasites

Mice infected with 2,000 Me49-FLUC tachyzoites were imaged 21 dayspost-challenge using the in vivo imaging system (IVIS; Xenogen, Alameda,Calif.). Mice were injected retroorbitally with 200 μl (15.4 mg/ml) ofD-luciferin, anesthetized in an O₂-rich induction chamber with 2%isoflurane, and imaged after 12 minutes. Photonic emissions wereassessed using Living image® 2.20.1 software (Xenogen). Data arepresented as pseudocolor representations of light intensity and meanphotons/region of interest (ROI). All mouse experiments were repeated atleast twice.

Enumeration of Cysts in Mouse Brains Following Type II ParasiteChallenge

Mice were euthanized at 21 days after infection with 10,000 ofMe49-Fluc, and brains were collected, homogenized with 1 ml of saline(0.85% NaCl). Tissue cysts were counted microscopically in 50 μl of thehomogenate, and the count was multiplied by 20 to obtain the number oftissue cysts per brain. This number was confirmed by staining braincysts with fluorescein-labeled Dolichos biflorus agglutinin (VectorLaboratories) and quantitation using fluorescence microscopy.

Flow Cytometry.

Splenocytes were manually processed using 70 μm filters in DMEM mediasupplemented with 5% FCS and red blood cells were lysed with ACK lysisbuffer. Cells were stained with CD3 APC (145-2C11), CD4 PE (GK1.5), CD8PerCP (53-6.7), and CD44 AF780 (IM7), CD45RB FITC (C363.16A). Allantibodies were purchased from eBioscience (San Diego, Calif.). Memory Tcells were defined as CD44^(hi)CD45RB^(lo). All flow cytometry data wascollected on LSRII flow cytometer (BD Biosciences, San Jose, Calif.) andanalyzed using FlowJo software 10.0 (Tree Star, Ashland, Oreg.).

Statistical Analyses

Data for each assay were compared by One-way ANOVA or a Student t testusing GraphPad Prism 5 software (GraphPad Software, San Diego, Calif.).Differences between the groups were identified by ANOVA and multiplecomparison procedures, as we previously described [7]. Data areexpressed as the means±SD. Results were considered to be statisticallysignificant at p<0.05.

Results

CD4 T Cell-Stimulating Peptide, AS15 with the Adjuvant GLA-SE (LipidTLR-4 Agonist) Resulted in an Increased IFN-γ Production.

Splenocytes were isolated from immunized HLA-A11 transgenic mice 24hours after the second immunization. Their ability of lymphocytes togenerate IFN-γ and proliferate in response to AS15 peptide was assessedusing ELISpot assay. The data (not shown)indicate IFN-γ production byAS15 peptide stimulation in vitro was significantly enhanced in miceimmunized with adjuvanted AS15 peptide compare to AS15 peptide alone.

AS15 with the Adjuvant GLA-SE Causes Storm of Cytokines Expression and TCell Marker, Correlated with Serious Defects in HLA A11 Transgenic MicePathology and Death.

There is a remarkable susceptibility of HLA-A11 transgenic mice to theimmunization with AS15 plus GLA-SE. At day 3-4 post-boost immunization,a majority of mice died. To better understand the fate of these miceafter second immunization and to avoid the loss of mice after secondimmunization, blood was collected and the cytokine levels in the serawere analyzed before and after 24 hours post immunization. There was asignificant increase of the level of IFN-γ, TNF α, and IL6, in the seraof immunized mice. Because, a majority of these mice died at day 3-4after boost-immunization, we examined at 24 h the cytokines levels inthe serum.

GLA-SE was found to induce a strong Th1 response that includes initialinduction of IL-12, which in turn stimulates IFN-γ production. Toconfirm that AS15 with the adjuvant GLA-SE treatment increased Th1cytokine production and to understand how the immunogene was affectingthe fate of mice, we measured serum levels of IFN-γ, IL-12p70, IL-2,IL-10, and IL-6 by a cytokine bead array at 24 hours before and aftersecond immunization (data not shown). When AS15 combined to GLA-SE,there is a significant increase of the level of IFN-γ, tumor necrosisfactor α, and interleukin 6 after 24 hours post-II immunization. We alsoevaluated whether weight loss and mortality in mice immunized withadjuvanted AS15 was associated with liver damage and/or damages in thegut during infection. Severe necrosis of the ilea, predominantly withinthe villi, was observed in treated mice. Using Caspase3 marker, wedemonstrated the presence of apoptotic cells in small intestine region(data not shown). In contrast, the ileum of untreated mice has a normaldistribution. This is correlated with increase of infiltration of CD3T-Cell. Changes also occur in the liver of treated mice.

Surprisingly, 5 out of 13 mice which survive the storm reduce parasiteburden when mice are infected with the virulent Toxoplasma type I. Moreinterestingly, there is a difference between the sex of mice in responseto the immunization by AS15 and adjuvant. Our results showed that maleHLA-A11 transgenic mice are more resistant to the boost immunization.None of mice (15 mice tested) died and all are resistant against deathafter infection.

Addition of HLA-A11-Restricted CD8⁺ T Cell Epitope Peptides and/or PADREAbrogates AS15 Phenotype.

Since IFN-γ and CD8⁺ T Cells have been shown to be critical for survivalof mice after infection with Toxoplasma, we sought to combine AS15peptide, PADRE and HLA-A11 restricted CD8⁺ peptides for more increase ofIFN-γ and better protection against T. gondii. As expected, immunizationwith AS15-GLA induced effective IFN-γ production from CD4 T cells (FIG.14A). Surprisingly, when PADRE helper epitope was added to AS15 plusGLA, it reduces the amount of IFN-γ production. In other hand when HLAA-11-restricted CD8⁺ T cell epitope peptides from GRA6 (AMLTAFFLR (SEQID NO: 18)), SAG1 (KSFDILPK (SEQ ID NO:)), SAG2C (STFWPCLLR(SEQ ID NO:14)), SPA (SSAYVFSVK (SEQ ID NO: 15)), SPA (AVVSLLRLLK (SEQ ID NO: 17))were added, robust CD8⁺ T cell production of IFN-γ was achieved (FIG.14B).

PADRE added to AS15 Plus GLA Protects Mice at Early Stage Against Type Iand II Parasite Challenge.

We immunized twice HLA-A11 mice with AS15-GLA, AS15-GLA-PADRE, CD8⁺ Tcell restricted peptides-PADRE-GLA, AS15-CD8⁺ T cell restrictedpeptides-PADRE. PBS or adjuvants alone were used as control. Wechallenged mice after 10 days with type I strain of T. gondii (2000 RHtachyzoites). Peritoneal fluid was collected 5 days post-infection andparasite fluorescence and numbers were measured using fluorometer andhemocytometer, respectively. Compared to control, fluorescence from allimmunized mice was significantly lower (FIG. 15A). This reduction wasalso observed in the measurements of the total parasite burden (data notshown). Most interestingly, immunized mice with AS15-GLA (n=8 mice) showserious defects in growth and death 3 days after the secondimmunization, however adding PADRE abrogates this phenotype. Thesurvived mice (n=3 mice) show the most protective protection against RHparasites. We decided to investigate in more details the response ofmice to AS15-GLA and not include this group in our survival analysiswith T gondii type II strain.

We next addressed whether the combination of epitopes that wereidentified could confer protection against Me49 type II parasitechallenge in HLA-A11 mice. Ten days after the second immunization, micewere challenged with 2000 Me49 (Fluc) that expresses the Fireflyluciferase (FLUC) gene and imaged the brain at 21 days using the in vivoimaging system. As shown in FIG. 15B, the number of luciferaseexpressing parasites in the immunized HLA-A11 mice was significantlyreduced compared to the unimmunized mice. The attenuated parasites byknockout of the ribosomal protein 13 gene (rps13Δ) were used as positivecontrol. This correlates with survival studies shown in FIG. 15C, whichshown a majority of mice immunized with the AS15-GLA-PADRE, AS15-CD8⁺ Tcell restricted peptides-PADRE-GLA survived parasite challenge. Incontrast, only 1 of 5 (20%) unimmunized mice survived parasitechallenge. CD4⁺ T cell and CD8⁺ T cell response deliver respectivelyprotection help at early and late stage after immunization.

The ability of AS15-GLA-PADRE and AS15-CD8⁺ T cell restrictedpeptides-PADRE-GLA to protect mice against T. gondii was evaluated. Micewere immunized with same groups and were the subject of challenge after10 or 35 days. As shown in FIG. 16, great protection after 35 days postimmunization was observed when HLA-A11 mice CD8⁺ T cell restrictedpeptides were added. This is in contrast with the response of thesepeptides after 10 days post immunization (FIG. 15A). PADRE has betteroutcome than AS15 plus GLA when added to CD8⁺ T cell to increase memoryCD8⁺ T Cell response

We then analyzed the effect of PADRE or AS15 added to CD8⁺ T cellpeptides on the T. gondii specific CD8⁺ T cell memory response. This wasperformed by quantifying the levels of memory T cells in the spleen fromT. gondii immunized 35 days after the last immunization. CD8⁺ memory Tcells was significantly increased with mice immunized with PADRE plusGLA- to CD8⁺ T cell peptides compared with the adjuvant alone or PBS(data not shown).

Discussion

We sought to combine AS15 peptide, PADRE and HLA A-11 restricted CD8⁺peptides for more increase of IFN-γ and better protection against T.gondii. Our data show help from CD8⁺ T cell peptides for increase ofIFN-γ expression. Interestingly, addition of PADRE, another restrictedCD4⁺ T Cell to AS15 peptide reduces the increase of IFN-γ from CD4⁺ TCell and abrogates AS15 phenotype.

We didn't see the presence of apoptotic cells or necrosis in liver andsmall intestine cells when PADRE was added. Furthermore, AS15 and PADREseems playing a protective role immediately after boost immunization.However, CD8⁺ T cell peptides help to protect mice after longimmunization period. This might be explained by the long lasting immuneresponse and contribution of CD8⁺ T cell restricted peptides to increasememory CD8⁺ T Cell response. It is not clear why PADRE abrogates theAS15 phenotype. Two hypotheses might explain this role. PADRE mightactivate T regulatory cells (Treg) to reduce the response induced byAS15 or PADRE is competing with AS15 for T Cell stimulation. To rule outthis possibility, we examined the PADRE and AS15 -specific CD4⁺ T cellsgenerated for their expression of FoxP3, a marker for CD4⁺ CD25⁺ Tregulatory cells. We found that there is no difference in the expressionof FoxP3 between the two groups, suggesting that the PADRE effect is notdue to the activation of CD4⁺CD25⁺ T regulatory cells (data not shown).Furthermore, we examined the T cell stimulation generated by AS15 orAS15+PADRE for their expression to CD107a and b, a marker for cytotoxicT cells. We found that some mice from the group stimulated with AS15presented high level of cd107a/b. This is in correlation with thevariation found in this group.

Example 5

“Tox-All” is another example of a SAPN. Tox-all follows the sameprinciple as the A11 SAPN (SEQ ID NO:27) but it has multiple componentsto stimulate diverse HLA haplotype CD8 T cells, CD4 T cells, B cells tomake antibody, and contains the TLR5 ligand flagellin. These areincluded to stimulate multiple arms of the immune response. This iscalled “Tox-All” to reflect all the components. The sequence of Tox-Allis:

(SEQ ID NO: 28) (MGDDHHHHHHHHHH)WFMGVLVNSLQDITMGSLFFVQDFMIVSISLVQDGLAAAVVAVQDLPQFATAATRDSPASGRYIQQMLDQRCQEIAAELCQSGLRKMCVPSSRIVARNAVGITHQNTLQWRCFDTASLLESNQENNGVNCVDDCGHTIPCPGGVHRQNSNHATRHEILSKLVEEGVQRFCSPYQASANKYCNDKFPGTIARRSKGFGNNVEVAWRCYEKASLLYSVYAECASNCGTTWYCPGGRRGTSTELDKRHYTEEEGIRQAIGSVDSPCSEVEVCLPKDENPPLCLDESGQISRGSWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWMGGRLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEELARGMAQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNVQQEYESDDAVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLRDSDSDTENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

The schematic diagrams of SAPN A11 (SEQ ID NO:27) in FIG. 17A 12e issimilar to the schematic diagram of the more complex SAPN Tox-All (FIG.17B). (FIG. 12f ). FIG. 17C shows that this Tox-All has the proper sizeand folding and purity of the desired SAPN. Tox-All contains CD8 T celleliciting epitopes by binding to HLA All, A2, and B7, PADRE a universalCD4 T cell eliciting epitope, protein domains that can elicit antibody(data not shown). Tox-All has the same scaffold flagellin that is shownfor SAPN A11. Tox-All and the data provided demonstrate that we canproduce and begin to build a multicomponent/epitope protein thatstimulates an immune response in HLA A2 and HLA A11 mice and antibody,and a response to the CD4 epitope PADRE. It also contains the TLR5adjuvant Flagellin and also stimulates TLR5 (data not shown). AdditionalCD8 peptides that bind to other HLA molecules to broaden populationcoverage and CD4 peptides can be added in this complex SAPN. Additionalproteins that elicit B cells can also be included in the SAPN. Thisallows us to tailor an immune response for each component of the immunesystem we wish to stimulate. The methods and approaches are thosedescribed for the A11 SAPN.

1. An isolated polynucleotide encoding a chimeric polypeptide, whereinthe chimeric polypeptide comprises (a) a plurality of first peptidedomains wherein each first peptide domain is a CD8⁺ T cell elicitingepitope, including but not limited to FLSLSLLVI (SEQ ID NO: 1),FMIAFISCFA (SEQ ID NO: 2), FVIFACNFV (SEQ ID NO: 3), FMIVSISLV (SEQ IDNO: 4), FLLGLLVHV (SEQ ID NO: 5), FLTDYIPGA (SEQ ID NO: 6), ITMGSLFFV(SEQ ID NO: 7), GLAAAVVAV (SEQ ID NO: 8), VLLPVLFGV (SEQ ID NO: 9),FAAAFFPAV (SEQ ID NO: 10), VVFVVFMGV (SEQ ID NO: 11), FMGVLVNSL (SEQ IDNO: 12), FLVPFVVFL (SEQ ID NO: 13), STFWPCLLR (SEQ ID NO: 14), SSAYVFSVK(SEQ ID NO: 15), KSFKDILPK (SEQ ID NO: 16), AVVSLLRLLK (SEQ ID NO: 17),and AMLTAFFLR (SEQ ID NO: 18)); and (b) one or more second peptidedomains, wherein each second peptide domain is a CD4+ epitope, includingbut not limited to AKFVAAWTLKAAA (SEQ ID NO: 19), AVEIHRPVPGTAPPS (SEQID NO: 20), IRLLASLHH (SEQ ID NO: 39), LIRLLASLH (SEQ ID NO: 40),LTLQLIRLL (SEQ ID NO: 41), VIEEFNRI (SEQ ID NO: 42), LQLIRLLAS (SEQ IDNO: 43), IDVVIEELF (SEQ ID NO: 44),or a derivative thereof.
 2. Theisolated polynucleotide of claim 1, wherein (i) the plurality of firstpeptide domains comprises at least 5 peptide domains, wherein the atleast 5 peptide domains include KSFKDILPK (SEQ ID NO: 16), STFWPCLLR(SEQ ID NO: 14), AVVSLLRLLK (SEQ ID NO: 17), SSAYVFSVK (SEQ ID NO: 15),AMLTAFFLR (SEQ ID NO: 18); and/or (ii) the one or more second peptidedomains include AKFVAAWTLKAAA (SEQ ID NO: 19) or a derivative thereof.3. (canceled)
 4. The isolated polynucleotide of claim 1, wherein some orall of the plurality of first peptide domains are separated from theother first peptide domains peptides by a spacer of 1 or more amino acidresidues and/or by the one or more second peptide domains.
 5. Theisolated polynucleotide of claim 4, wherein the spacer is selected fromthe group consisting of N, K, NA₁₋₃(SEQ ID NO: 21), KA₁₋₃(SEQ ID NO:22), and GPGPG (SEQ ID NO: 23).
 6. The isolated polynucleotide of claim1, wherein the chimeric polypeptide comprises the amino acid sequence(SEQ ID NO: 24) AVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLR


7. The isolated polynucleotide of claim 1, wherein the chimericpolypeptide further comprises a third peptide domain comprising apeptide capable of promoting self-assembly/multimerization (2, 3, 4, 5,6, or more assembled copies) of the polypeptide.
 8. (canceled)
 9. Theisolated polynucleotide of claim 7, wherein the third peptide domaincomprises the sequence selected from the group consisting of: (SEQ IDNO: 25) WEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWM; (SEQ ID NO: 26)RLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEE L;

or both.
 10. The isolated polynucleotide of claim 7, wherein thechimeric polypeptide comprises the amino acid sequence SEQ ID NOS:27-28.A11 (residues in parentheses are optional) (SEQ ID NO: 27)((MGDKHHHHHHHHHH))KDGSDKGSWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWMGGRLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEELARGMAQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNVQQEYESDDAVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLRDSDSDTENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQA GTSVLAQANQVPQNVLSLLR

Tox-all (residues in parentheses are optional) (SEQ ID NO: 28)(MGDDHHHHHHHHHH)WFMGVLVNSLQDITMGSLFFVQDFMIVSISLVQDGLAAAVVAVQDLPQFATAATRDSPASGRYIQQMLDQRCQEIAAELCQSGLRKMCVPSSRIVARNAVGITHQNTLQWRCFDTASLLESNQENNGVNCVDDCGHTIPCPGGVHRQNSNHATRHEILSKLVEEGVQRFCSPYQASANKYCNDKFPGTIARRSKGFGNNVEVAWRCYEKASLLYSVYAECASNCGTTWYCPGGRRGTSTELDKRHYTEEEGIRQAIGSVDSPCSEVEVCLPKDENPPLCLDESGQISRGSWEEWNARWDEWENDWNDWREDWQAWRDDWARWRATWMGGRLLSRLERLERRNEELRRLLQLIRHENRMVLQFVRALSMQNAELERRLEELARGMAQVINTNSLSLLTQNNLNRSQSALGTAIERLSSGLRINSARDDAAGQAIANRFTANIRGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVRVLAQDNTLTIQVGANDGETIDIDLRQINSQTLGLDQLNVQQEYESDDAVVSLLRLLKNAMLTAFFLRNAAAKSFKDILPKKAAASSAYVFSVKKAAAKFVAAWTLKAAAKSTFWPCLLRDSDSDTENPLQRIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR


11. The isolated polynucleotide of claim 1, wherein the chimericpolypeptide further comprises a secretory signal, including but notlimited to the murine Igκ-chain signal sequence.
 12. The isolatedpolynucleotide of claim 11, wherein the secretory signal is present atthe N-terminus of the chimeric polypeptide.
 13. A recombinant expressionvector, comprising the isolated polynucleotide of claim 1 operativelylinked to a control sequence.
 14. (canceled)
 15. The recombinantexpression vector of claim 13, wherein the vector comprises a geneencoding a self-amplifying RNA operatively linked to the polynucleotide.16. The recombinant expression vector of claim 15, wherein theself-amplifying RNA comprises the nucleic acid sequence of SEQ ID NO:29(SEQ ID NO: 29) GAAUUUGCUGCGACGAUGGGUAUGCAAGUCCAGAUCCAGAGCCUGUUCCUGCUGCUGCUGUGGGUGCCGGGUUCACGCGGUAUGGCGGUGGUUAGCCUGCUGCGUCUGCUGAAAAACGCCAUGCUGACCGCAUUUUUCCUGCGCAAUGCGGCCGCAAAGAGUUUCAAGGAUAUCCUGCCGAAAAAGGCUGCGGCCAGCUCUGCGUAUGUCUUUUCCGUGAAAAAGGCAGCUGCGAAAUUCGUUGCUGCCUGGACCCUGAAAGCUGCCGCUAAAUCGACGUUCUGGCCGUGUCUGCUGCG U.


17. The recombinant expression vector of claim 15, wherein theself-amplifying RNA is encoded by the nucleic acid sequence of SEQ IDNO:30. (SEQ ID NO: 30)CATGGGTATGCAGGTCCAGATTCAGTCACTCTTTCTCCTCCTCCTCTGGGTCCCCGGTAGCCGGGGTATGGCCGTGGTCAGCCTGCTCAGGCTGCTCAAGAACGCCATGCTGACCGCTTTCTTTCTCAGAAATGCCGCTGCAAAGTCTTTCAAAGACATCCTGCCCAAGAAAGCCGCTGCAAGCTCCGCCTACGTGTTCAGTGTCAAGAAAGCCGCTGCAAAATTTGTGGCC


18. A chimeric polypeptide comprising the chimeric polypeptide encodedby the polynucleotide of claim
 1. 19. (canceled)
 20. A chimeric RNA,comprising the RNA expression product of the polynucleotide claim
 1. 21.(canceled)
 22. A pharmaceutical composition, comprising: (a) thechimeric polynucleotide of claim 1; and (b) a pharmaceuticallyacceptable carrier.
 23. The pharmaceutical composition of claim 22,wherein composition comprises an adjuvant, such as a TLR4 ligand.24.-26. (canceled)
 27. The pharmaceutical composition of claim 22,wherein the composition comprises a vaccine, such as a vaccineformulated for injection or electroporation.
 28. A method for treatingor limiting Toxoplasma gondii infection, comprising administering to asubject at risk of Toxoplasma gondii infection an amount effective totreat or limit the infection of the chimeric polynucleotide of claim 1.29.-31. (canceled)